Method for fixing dna-binding proteins in cells, and washing liquid

Abstract

The invention relates to a method (100) for fixing DNA-binding proteins (5.2) in cells. The method (100) comprises: a) providing (10) target cells (2); b) irradiating (11) the target cells (2) with UV rays (3) having a wavelength in the range from 240 nm to 280 nm and an energy density in the range from at least 0.01 mJ / cm2 to 1000 mJ / cm2; and c) incubating (12) the target cells (2) in a washing liquid (200) and washing out non-DNA-bound proteins from the target cells (2). The invention also relates to a washing liquid (200) which can be used, for example, in such a method (100).

Description

[0001] Method for fixation of DNA-binding proteins in cells and washing fluid

[0002] The invention relates to a method for fixing DNA-binding proteins in cells, in particular single cells of a cell mixture, as well as to a washing liquid and its use.

[0003] Nucleic acids, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), carry genetic information in all living organisms and viruses. The reading, maintenance, and replication of nucleic acids require specialized cellular proteins that bind directly to the nucleic acids and interact functionally with them. More than two thousand different DNA-binding proteins have been described for humans, accounting for approximately 10% of all human proteins. However, each cell expresses only the portion of DNA-binding proteins required for its specific cellular function, and the binding of proteins to nucleic acids is strictly regulated. A portion of the DNA-binding proteins is present in cells in an inactive, non-DNA-bound form.Only after cell stimulation, for example, through altered hormone secretion or external factors, do some proteins undergo functional activation and DNA binding. Cells thus possess a specific DNA-protein binding pattern depending on the cell type and activation state. Dysregulation or mutations of DNA-binding proteins often trigger pathogenic changes and can be used both as markers for disease identification and prognosis and as therapeutic targets.

[0004] Nucleic acid-binding proteins must generally bind directly to the nucleic acid to fulfill their cellular functions. However, there is often an excess of the DNA-binding protein under investigation in the cell plasma or nucleus, and only a portion of the protein or only a specific subtype of the protein (e.g., due to post-translational modifications) actually binds to the DNA and is functional. To date, the in vivo determination of the proteins that actually interact with DNA in individual cells or cell mixtures has not been possible. Current high-throughput methods, such as the intracellular staining of proteins after formaldehyde fixation, only allow the detection of the total amount of a specific DNA-binding protein in the single cell and cannot distinguish whether the protein was actually bound to DNA at the time of analysis.

[0005] In addition, existing methods have only a low temporal resolution. For example, previously used chemical fixation methods require a reaction time of several minutes.

[0006] Another disadvantage of fixation with formaldehyde and other fixatives is the blocking of antibody binding sites by their binding to proteins. This makes many antibodies unusable for protein detection. Therefore, specific antibodies must be produced and tested. Currently, there are only suitable antibodies for a few transcription factors (max. 100). Antibody production represents a large market, as there are currently no alternative native methods for detecting / labeling proteins.

[0007] The introduction and release of substances into and from cells has been described in various places.

[0008] For example, US 2005 / 095578 ​​A1 describes methods and devices for introducing and releasing substances into and from cells, and in particular methods and devices for temporarily permeabilizing a living cell so that one or more of a variety of substances, such as ions, proteins and nucleic acids, can be loaded into or released from the cell.

[0009] WO 2009 / 140701 A2 describes the use of ultrashort pulse (USP) lasers for introducing macromolecules into cells. It presents methods for introducing macromolecules such as proteins, peptides, amino acids, nucleic acids, DNA, RNA, oligonucleotides, lipids, carbohydrates, or any combination thereof into cells with cell walls, such as bacteria, archaea, fungi, yeast, algae, and plant cells, using an ultraviolet USP laser.

[0010] Specific, direct DNA-protein interactions cannot currently be quantified at the single-cell level because existing methods, such as intracellular staining of proteins after formaldehyde fixation, cannot distinguish between DNA-bound (also called transcription factors) and DNA-unbound proteins.

[0011] In the publication by REIM, A. et al., "Atomic-resolution mapping of transcription factor-DNA interactions by femtosecond laser crosslinking and mass spectrometry." Nat. Commun. 2020, 11 (1):3019, printed pages 1-14, target cells are irradiated with pulsed UV laser radiation at 258 nm, which generates protein-DNA complexes and increases the cross-linking rate between DNA and proteins. The samples are then denatured, using, among other things, a wash buffer to purify protein-DNA fragments. The fragments of the protein-DNA complexes are then analyzed by mass spectrometry.

[0012] A publication by Nizamuddin, S. et al., "Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT&RUN." Nucleic Acids Res. 2021, 49 (9):e49, Print Page 1-18, discloses a buffer containing, among other substances, digitonin for permeabilizing the nuclear membrane.

[0013] The invention is based on the object of providing a method that enables the fixation of DNA-binding proteins in cells without destroying the cells. Furthermore, a buffer that can be used in such a method is to be provided.

[0014] The problem is solved by the features of the independent patent claims. Further useful embodiments of the invention are the subject of the dependent patent claims.

[0015] To solve this problem, a method for immobilizing DNA-binding proteins in cells, particularly single cells of a cell mixture, is proposed. For example, the method can be used to detect and quantify DNA-bound proteins in such cells.

[0016] The proposed method comprises the following steps: a) providing target cells, b) irradiating the target cells with UV rays of a wavelength in the range of 240 nm to 280 nm and an energy density in the range of 0.01 mJ / cm 2 up to 1000 mJ / cm 2 , c) incubating the target cells in a washing liquid and washing out non-DNA-bound proteins, in particular proteins not directly bound to DNA, from the target cells.

[0017] In process step c), proteins that are not directly bound to DNA can be washed out. Preferably, proteins that potentially bind DNA, i.e., proteins that could in principle bind to DNA, but were not directly bound to DNA during irradiation in process step b), can be washed out. In other words, the directly DNA-bound proteins can be isolated in the intact cell.

[0018] The method is performed outside the human or animal body. Optionally, the method may include detecting covalently DNA-bound proteins in the target cells. Such detection may, for example, include quantifying the covalently DNA-bound proteins. The term "in the target cells" here means that the target cells are not lysed, i.e., the detection occurs in the intact target cells. This requires that no lysis occurs in the previous process steps, i.e., the target cells remain intact.

[0019] For detection, the fixed DNA-bound proteins can be stained, e.g., using labeled antibodies. In this case, the stainability can be influenced by UV irradiation and the use of the washing liquid, for example, by non-masking, covalent bonds to other regions of the genome that may be inaccessible by chemicals, e.g., the dye, and / or staining in other buffers. In contrast to chemical fixation, the staining or stainability after UV fixation / crosslinking can be altered because, unlike chemicals such as formaldehyde, which can surround the antibody binding site (masking), UV irradiation leaves no residue. In addition, UV irradiation sometimes generates protein-DNA crosslinks in genome regions that may be inaccessible to chemicals. This can also alter the staining or stainability.

[0020] The proposed method allows for the formation of covalent DNA-protein bonds inside the target cell while preserving the target cells, i.e., without lysis. Furthermore, remaining non-DNA-bound proteins, especially excess potentially DNA-binding proteins, can be washed out, allowing subsequent detection of the DNA-protein bonds in the intact cell. Advantageously, all non-DNA-bound proteins can be completely washed out to improve the accuracy of subsequent detection.

[0021] Particularly advantageously, the method allows the detection of exclusively DNA-bound proteins in single cells of a cell mixture.

[0022] The term "target cells" refers to cells in which DNA-binding proteins can be fixed and isolated, and optionally detected and quantified, according to the proposed method. The target cells provided according to step a) can be homogeneous or heterogeneous single cells, or a cell mixture or cell cluster. For example, the target cells can be isolated, i.e., in the form of single cells, or as a cell mixture, whereby several different cell types can also be present in the cell mixture.

[0023] The proposed method is applicable to all cell types. For example, the target cells can be animal cells, plant cells, eukaryotic cells, or prokaryotic cells. Preferably, the target cells can be cells of metazoans or higher, i.e., more complex, organisms or multicellular organisms. Depending on the cell type, the process parameters can vary. For example, the energy density to be selected can depend on the cell type. For example, a high energy density may be necessary for plant cells, while a low energy density, e.g., in the range between 0.01 and 1 mJ / cm 2 , may be sufficient. The washing fluid can also be selected depending on the cell type.

[0024] The target cells can be provided in a liquid so that the irradiation of a cell suspension can take place in process step b). The liquid can therefore also be referred to as the irradiation liquid. The liquid should preferably be isotonic, i.e. such that a cell dispersed in the liquid does not change its volume. Furthermore, the absorption of the UV rays used in process step b) should be as low as possible, e.g. below 10%, preferably below 5% and more preferably below 1%. A phosphate-buffered saline solution (PBS), for example, can be used as the liquid. A medium with or without indicator dye can be used as the liquid, e.g. Dulbecco's Modified Eagle's Medium (DMEM) without phenol red, RPMI-1640 without phenol red, Iscove's Modified Dulbecco's Medium (IMDM) without phenol red.

[0025] The term “washing liquid” is understood here to mean a liquid, e.g. a mixture of substances in liquid form, e.g. as an aqueous solution. The washing liquid is preferably an isotonic saline solution that maintains the integrity of the cells, i.e. a cell-preserving solution. The washing liquid can be identical to the liquid in which the target cells are provided and irradiated, i.e. the washing liquid can also serve as the irradiation liquid. If the washing liquid is not identical to the irradiation liquid, the washing liquid is added to the target cells after irradiation to carry out method step c). Optionally, the irradiation liquid can be completely or partially replaced with the washing liquid.

[0026] The washing liquid can be a buffer solution or buffered liquid, i.e., a liquid whose pH changes significantly less upon addition of an acid or base than would be the case with an unbuffered system. For example, the washing liquid can be a wash buffer, incubation buffer, or a similarly suitable buffer. The washing liquid can be based on, for example, a phosphate-buffered saline solution (PBS). Alternatively, the washing liquid can be Tyrode's solution, balanced salt solution (BSS), or Ringer's solution. The pH of the washing liquid can be, for example, 7.4. Its property as a buffer solution enables work at this constant pH.

[0027] The term "energy density" refers to a measure of the energy input per area of ​​the cell(s). Energy density can be determined, for example, based on the number of photons that hit the cell surface in a laser pulse, e.g., 5 ns. This can be measured using a sensor for measuring the applied energy, which is positioned in the laser beam instead of the sample. Since the laser beam is not focused and the medium does not absorb the laser radiation, the energy density at the measuring device corresponds to the energy density on the target cells. For example, irradiating the target cells with an energy density of 10 mJ / cm 2 in process step b) that in 1 cm 2 10 mJ of energy is applied to the cell surface. For this purpose, the laser power and / or the irradiation duration, e.g., pulse duration and / or pulse number, can be varied accordingly.

[0028] The proposed method is based on two properties of DNA:

[0029] 1. the photochemical activation of nucleic acids by UV radiation, which leads to the formation of covalent bonds with attached proteins and

[0030] 2. the fact that, due to its enormous molecular size, DNA cannot leave the nucleus after cell permeabilization, thus fixing the covalently bound proteins in the cell for subsequent detection reactions. Proteins that are not present in the immediate vicinity of the DNA at the time of irradiation are subsequently removed as soluble components in washing steps and therefore cannot be detected.

[0031] Irradiation of the target cells with high-intensity UV light, e.g., UV laser light, leads to the formation of covalent bonds between proteins already bound to the DNA and the DNA. For the purposes of this invention, a covalent bond is understood to be a form of chemical bonding with bonding electron pairs, which ensures the strong cohesion of atoms in molecularly structured chemical compounds.

[0032] In contrast, the use of conventional chemical fixation agents, such as formaldehyde, creates non-selective cross-links between proteins and protein nucleic acids in the cell, making the detection of direct protein-nucleic acid interactions impossible. In the irradiation carried out according to step b) of the proposed method, instead of non-selective cross-links, photochemical cross-linking is induced by high-intensity UV radiation, which only creates cross-links between protein and nucleic acid molecules that are in direct contact, i.e., a single DNA base is cross-linked with an amino acid that is in direct contact (distance in the angstrom range). In contrast, chemical fixation leads to the non-specific covalent binding of proteins to structures throughout the cell and / or to other proteins.Secondly, the cell membranes of the target cells are damaged by the irradiation and thus become porous, e.g. permeable to dye molecules. The irradiation, together with the washing liquid, causes effective pore formation, whereby non-covalently cross-linked proteins can be washed out more easily. Consequently, proteins not bound to DNA can be washed out during subsequent incubation of the target cells in the washing liquid. Since the DNA of a cell in the nucleus is too large to be washed out of the cell, this DNA and all proteins covalently bound to it by UV treatment remain in the cell. There they can be labeled or stained and subsequently analyzed, for example, using low- and high-throughput methods. Both proteins not bound to DNA and proteins indirectly bound to DNA, i.e. proteins that are only bound to the DNA via other proteins, are washed out.The reason for this is that the photochemical reaction underlying the formation of a covalent bond originates from the DNA.

[0033] Advantageously, only proteins that were directly bound to the DNA in the living cells at the time of irradiation remain in the cells. These proteins are available for follow-up studies. The detection of multiple target proteins can create a "fingerprint" characteristic of the cell, which can be used, for example, for diagnostic purposes.

[0034] Preferably, the method is carried out in such a way that the covalent bonding of DNA with proteins is carried out in combination with the washing out of all potentially DNA-binding, but not covalently fixed to DNA, proteins from the target cells.

[0035] It is advantageous that the cells remain intact, especially externally intact, during the induction of DNA-protein binding. This means, within the context of the invention, that the cells are not lysed and, in particular, that the integrity of the cells is maintained and the epitopes of the proteins to be detected are not damaged.

[0036] The irradiation of the target cells according to process step b) and the incubation of the target cells and washing out according to process step c) can preferably take place simultaneously or at least immediately following one another. Advantageously, the effect of the washing liquid can already develop during the irradiation, and the fixation of the DNA-binding proteins can be carried out in a time-saving manner. According to various embodiments, the target cells can be irradiated with pulsed UV laser radiation. Advantageously, this can increase the achievable energy density, particularly of low-power, i.e., weak lasers, when treating a few or individual cells using microfluidics concepts. When using a relatively weak laser, short pulses with a low frequency can preferably be selected so that the individual pulses achieve the necessary energy density.

[0037] Alternatively, a non-pulsed laser can be used to irradiate the target cells.

[0038] According to further embodiments, the irradiation can be carried out in such a way that the majority of the irradiated cells are irradiated exactly once.

[0039] Preferably, each cell should be hit by only one pulse, because crosslinking occurs in fs and a subsequent pulse could break existing crosslinks. For example, a laser pulse can provide an energy of approximately 50 mJ, last approximately 5 ns, and cover an area of ​​approximately 0.3 cm 2 The energy density can be achieved by reducing the area, increasing the energy, or shortening the pulse duration. The main reason is that two photons must hit the same spot very quickly one after the other to enable crosslinking. The higher the photon density, the higher the probability of this.

[0040] This avoids multiple irradiation and the resulting renewed destruction of the previously generated DNA-protein bonds.

[0041] To assess the frequency of irradiation, the cells can be stained with a dye that only fluoresces after irradiation. Rhodamine, for example, caged rhodamine, can be used as a dye for this purpose.

[0042] For the purposes of the invention, the phrase “irradiated once” means that the irradiated cell is hit by a laser exactly once. This can include a laser pulse or a very short irradiation with an unpulsed laser. It should be noted that a single irradiation is optimal and multiple irradiations reduce efficiency but do not prevent effectiveness. A single irradiation of the cells can, for example, be achieved by irradiating the cells in a suitable vessel, for example a tube, with simultaneous mixing, in particular by a stirrer. A person skilled in the art is able to adjust the cell concentration and the irradiation duration such that the majority of the cells are hit by a laser beam only once. Exemplary parameters to be selected for a cell concentration of 10 million would beCells in 200 ml of liquid are irradiated for 5 seconds at a pulse frequency of 10 Hz (equivalent to 50 pulses) at a stirring frequency of 500 rpm.

[0043] Alternatively, the type of irradiation can be influenced using microfluidics. For example, the cell flow rate can be adjusted so that each cell is hit by exactly one laser pulse.

[0044] The energy density to be achieved for the proposed process is in the range of 0.01 mJ / cm 2 up to 1000 mJ / cm 2 , e.g. 5 mJ / cm 2 up to 1000 mJ / cm 2 , preferably 10 mJ / cm 2 up to 1000 mJ / cm 2 , more preferably 20 mJ / cm 2 up to 500 mJ / cm 2 , particularly preferably 40 mJ / cm 2 up to 250 mJ / cm 2 , is influenced by various factors, such as pulse duration and pulse rate.

[0045] The pulse duration can preferably be in the range from femtoseconds to picoseconds to a few nanoseconds, whereby a pulse duration in the range of picoseconds to a few nanoseconds can achieve higher efficiency than a pulse duration in the femtosecond range. The lower the energy of the laser used, the shorter the pulse duration should be. The pulse frequency can preferably be in the range of a few hertz to a few megahertz or gigahertz with a pulse duration in the femtosecond range.

[0046] By using a relatively long pulse duration in the range of picoseconds to nanoseconds, crosslinking, i.e. the connection of DNA with proteins, is particularly efficient while at the same time the cells remain intact.

[0047] The lower the energy density during irradiation, the longer the exposure time should be.

[0048] For example, it may be provided that the irradiation of the target cells takes place with an irradiation duration in the range of a few milliseconds to a few seconds.

[0049] Advantageously, the relatively short fixation time allows for high-resolution temporal studies, whereas chemical fixation takes between 5 and 60 minutes, which can lead to protein binding changes during this time, and rapid DNA-protein binding changes cannot be measured. The proposed method creates a snapshot of DNA-protein binding in living cells (although the cells are, of course, only alive until the time of irradiation, but the state of DNA-protein binding corresponds to the state in the living cells).

[0050] In addition to parameters such as laser power, pulse duration, pulse frequency, and irradiation time, the energy density to be achieved can also be influenced, for example, by the light-optical and / or electron-optical systems of a device used for the proposed method. For example, the use of an optical system with optically active elements, e.g., lenses, mirrors, etc., such as a lens system with one or more lenses, can be provided to increase the photon density of a weaker laser.

[0051] According to further embodiments, a UV laser in combination with at least one light-optical and / or electron-optical system can be used to irradiate the target cells.

[0052] The light-optical and / or electron-optical system may preferably be a device for increasing the photon density, for example a lens, a mirror and / or a prism for deflecting the light beam.

[0053] It can further be provided that cell separation is carried out to provide the target cells, particularly in combination with the use of lower-energy lasers. This can advantageously ensure that each individual target cell is hit by laser radiation with the required energy density. Cell separation can be carried out, for example, using microfluidic methods. Here, the cells can be irradiated one after the other in a channel, e.g., made of UV-permeable quartz glass. According to various embodiments, it can be provided that the washing liquid contains at least one cholesterol-dissolving and / or pore-inducing substance.

[0054] Examples of cholesterol-dissolving and / or pore-inducing substances are saponins, such as digitonin and / or polyoxyethylene 20-sorbitan monolaurate, also known under the trade name Tween® 20. Products from the Leucoperm product line can also be used. Other cholesterol-dissolving and / or pore-inducing substances are also conceivable and can be used within the scope of the invention.

[0055] Advantageously, the use of cholesterol-dissolving and / or pore-inducing substances further increases the permeabilization of the cell and nuclear membranes, which was already induced by UV irradiation. Pores can be formed in the cell and nuclear membranes that are large enough to allow protein complexes not bound to DNA to pass through, allowing them to be more easily washed out of the cell. At the same time, the cells remain intact, and lysis does not occur. Pores with a size of 8 to 10 nm are preferentially formed.

[0056] The buffer may further contain a component that assists in the leaching of proteins. These are preferably non-ionic detergents or surfactants, such as octoxinol 9 (CAS number 9002-93-1), known, for example, as NP-40, Nonidet™ P40, or Triton™ X-100.

[0057] The wash fluid can optionally contain enzymes or inhibitors. For example, the wash fluid can contain ribonucleases (RNases), which digest RNA and also wash it out. Enzymes, such as protease inhibitors, can be added to prevent alteration of the proteins to be detected.

[0058] According to further embodiments, the washing liquid may further comprise components that enable the dissolution of non-covalent DNA-protein bonds and / or protein-protein bonds. Such a component is, for example, a salt, preferably an inorganic salt, such as a metal chloride.

[0059] Metal chlorides are characterized by high solubility in aqueous media, e.g. the buffer medium.

[0060] The salt can, for example, be selected from a group comprising NaCl, LiCl, KCl, MgCh, and CaCh. In other words, NaCl, LiCl, KCl, MgCh, and / or CaCh can be used as the salt. NaCl and / or LiCl are preferred.

[0061] For example, the salt can be present in the washing fluid at a higher, non-physiological concentration. The salt concentration in the washing fluid can be in the range of 300 mM to 700 mM, for example, in the range of 450 mM to 550 mM. The salt concentration can be, for example, 550 mM.

[0062] One function of salt can be to disrupt the interaction between DNA and protein. The more tightly the protein binds to the DNA, e.g., the more DNA binding domains the protein has, the higher the salt concentration should be. The inventors of the present invention assume that histones cannot be detached from DNA.

[0063] This higher, non-physiological concentration can improve the resolution of non-covalent DNA-protein bonds and / or protein-protein bonds, which should not be detected by the proposed method, so that the accuracy of the detection of covalently DNA-bound proteins in the target cells can be increased.

[0064] Alternatively, the salt can be present in the wash liquid at a lower concentration. The salt concentration in the wash liquid can range from 50 mM to 100 mM. For example, the salt concentration can be 75 mM.

[0065] This lower concentration can enable a gentle dissolution of the aforementioned bonds. According to various embodiments, the target cells can be incubated in the wash fluid while continuously moving the cells, for example, by rotation and / or shaking. This ensures that each individual cell comes into sufficient contact with the wash fluid's constituents. The incubation time can also be reduced.

[0066] According to further embodiments, the target cells can be incubated in the washing liquid for a period of 5 to 60 minutes. The incubation duration can be selected depending on the respective target protein(s), i.e., the proteins that are to be bound to the DNA. The incubation time can depend, for example, on the binding strength of the protein to the DNA. Proteins with many DNA-binding domains should be incubated or washed for longer.

[0067] According to various embodiments, at least individual process steps of the proposed method can be carried out in a multiwell plate or in or on a microfluidic component.

[0068] For the purposes of the invention, the term "microfluidic component" refers to a component suitable for carrying out individual process steps in the field of microfluidics, such as a chip laboratory. In its simplest form, the microfluidic component consists of a first reservoir, a channel, and a second reservoir. The cells are pumped through the channel from the first reservoir to the second reservoir and irradiated in the process.

[0069] According to further embodiments, the irradiation of the target cells can take place at a temperature of maximum 10 °C, preferably maximum 4 °C.

[0070] Temperatures should always remain below 42 °C to prevent protein denaturation. Lower temperatures (< 10 °C) are preferable because they reduce or inhibit any enzyme activity, such as proteases or nucleases. The lowest possible temperature is the freezing point of the liquid used.

[0071] By performing the irradiation at low temperature, damage to the cell membrane and the proteins to be detected can be reduced.

[0072] According to further embodiments, the detection of the covalently DNA-bound proteins may comprise adding at least one detection reagent to the irradiated target cells, wherein the detection reagent interacts with the covalently DNA-bound proteins.

[0073] In other words, for the detection of DNA-bound proteins in the intact target cells, after incubating the target cells in the washing liquid, at least one detection reagent can be added to the irradiated target cells, whereby the detection reagent interacts with proteins covalently bound to the DNA.

[0074] The detection reagent can contain at least one fluorescently labeled antibody. In addition to fluorescently labeled antibodies, other examples of detection reagents include metal-labeled antibodies, enzyme-labeled antibodies, or protein tag detection agents. Furthermore, the analyte can be detected using an unlabeled primary antibody in combination with a labeled secondary antibody. The addition of a specific detection reagent is not necessary if the protein to be detected is already labeled prior to fixation (e.g., fluorescently labeled cell proteins).

[0075] The proteins covalently bound to the DNA by UV light remain in their native conformation and can thus be detected by antibodies that recognize the native protein.

[0076] Advantageously, the prior permeabilization of the cells allows the detection reagents to penetrate the cells better.

[0077] It can further be provided that after the addition of at least one detection reagent, the detection of an interaction between the detection reagent and the proteins covalently bound to the DNA takes place, preferably by means of flow cytometry and / or microscopy and / or other single-cell analysis methods.

[0078] Preferably, the detection of the interaction can be achieved using a high-throughput method. Flow cytometry can be advantageously used in high-throughput applications, while microscopy can be used in low-throughput applications. Standard flow cytometers only measure total fluorescence. Microscopy, on the other hand, can advantageously verify the localization of proteins. Furthermore, background staining can be better eliminated with microscopy.

[0079] A further aspect of the invention relates to a washing liquid. The washing liquid comprises at least one cholesterol-dissolving and / or pore-inducing substance, at least one non-ionic detergent, and at least one salt. The washing liquid can be used for incubating cells, for permeabilizing cell membranes and / or nuclear membranes, and for extracting non-DNA-bound proteins from cells, for example, in one of the methods explained above. Consequently, the explanations of the method also serve to describe the washing liquid, and the advantages of the method are correspondingly associated with the washing liquid.

[0080] A mild detergent is preferably used as a nonionic detergent. Compared to harsh detergents, mild detergents have lower surfactant activity, are less denaturing, and better preserve protein structure.

[0081] Advantageously, the washing fluid preserves the integrity of the cells, washes out unfixed proteins and enables intracellular protein staining.

[0082] The cholesterol-dissolving and / or pore-inducing substance can increase the permeabilization of the cell and nuclear membranes. Pores can be formed in the cell and nuclear membranes that are large enough to allow protein complexes not bound to DNA to pass through, allowing them to be washed out of the cell. At the same time, the cells remain intact, and lysis does not occur.

[0083] According to various embodiments, the washing liquid can contain polyoxyethylene(20) sorbitan monolaurate and / or a saponin, preferably digitonin, and / or leucoperm as cholesterol-dissolving and / or pore-inducing substance.

[0084] The non-ionic detergent can assist in the leaching of unbound proteins. The non-ionic detergent can be, for example, Octoxinol 9.

[0085] The salt present in the proposed washing liquid can improve the dissolution of non-covalent DNA-protein bonds and / or protein-protein bonds.

[0086] The salt can, for example, be selected from a group comprising NaCl, LiCl, KCl, MgCh, and CaCh. In other words, NaCl, LiCl, KCl, MgCh, and / or CaCh can be used as the salt. NaCl and / or LiCl are preferred.

[0087] For example, the salt can be present in the washing fluid at a higher, non-physiological concentration. The salt concentration in the washing fluid can be in the range of 300 mM to 700 mM, for example, in the range of 450 mM to 550 mM. The salt concentration can be, for example, 550 mM.

[0088] Alternatively, the salt can be present in the wash liquid at a lower concentration. The salt concentration in the wash liquid can range from 50 mM to 100 mM. For example, the salt concentration can be 75 mM.

[0089] Advantageously, the invention enables, for the first time, the exclusive quantification of proteins directly bound to DNA in individual cells of a cell mixture. The analysis is characterized by its high simplicity and minimal time and resource expenditure. Furthermore, the proposed method allows the simultaneous investigation of multiple DNA-binding proteins per single cell, enabling the detailed characterization of cell populations.

[0090] The UV laser radiation does not change the binding sites for antibodies on proteins, so that the proposed method enables the use of many otherwise unusable antibodies against the native forms of the proteins and thus also improved detection.

[0091] The irradiated cells become porous and can be permeabilized and stained more quickly and easily. Penetration and staining of intracellular proteins by color-labeled antibodies is improved, in contrast to chemical fixation, which makes the cells difficult to permeabilize.

[0092] Another key advantage of the proposed method is that it simplifies automation. For chemical fixation, chemicals must be added to the cells, and the reaction must be stopped by adding other chemicals. Laser-induced optical fixation eliminates the need for the addition and removal of substances.

[0093] The technique can be used in combination with other methods to determine cellular parameters such as surface markers or other non-leachable proteins, lipids or nucleic acids.

[0094] The detection of direct DNA binding of proteins in high-throughput single-cell analysis is currently not possible using alternative methods. Measurement methods such as intracellular staining of post-translationally modified DNA-binding proteins cannot detect direct DNA binding and require modification-specific antibodies, which are only available for a small number of target proteins. The proposed method does not require antibodies against specific protein modifications to detect DNA binding and can therefore potentially be used to investigate all DNA-binding proteins for which specific detection reagents (e.g., antibodies) are available. This measurement approach thus targets a broad spectrum of potential objects of investigation in cell and molecular biology research.

[0095] The invention will be explained in more detail below with reference to exemplary embodiments and the accompanying drawings.

[0096] Fig. 1: a simplified flow chart of an exemplary method;

[0097] Fig. 2: a schematic representation of the sequence of an exemplary method;

[0098] Fig. 3: an exemplary experimental setup for carrying out an exemplary method;

[0099] Fig. 4: the graphical representation of the results of flow cytometry after performing the exemplary method; and

[0100] Fig. 5: an illustration of microfluidics.

[0101] Figure 1 shows a simplified flow diagram of an exemplary method 100 for immobilizing DNA-binding proteins in cells. In a first step 10, target cells are provided. In the subsequent method step 11, the provided target cells are irradiated with UV rays having a wavelength in the range of 240 nm to 280 nm, preferably in the range of 250 nm to 270 nm, and an energy density in the range of 0.1 mJ / cm 2 up to 1000 mJ / cm 2 , for example 5 mJ / cm 2 up to 1000 mJ / cm 2 or 10 mJ / cm 2 up to 1000 mJ / cm 2 , preferably 20 mJ / cm 2 up to 500 mJ / cm 2 , particularly preferably 40 mJ / cm 2 up to 250 mJ / cm 2, resulting in covalent cross-links between DNA and DNA-bound proteins. The target cells remain intact during irradiation. In process step 12, the intact target cells are incubated in a washing liquid 200, which (further) permeabilizes the nuclear membrane and the cell membrane and washes non-DNA-bound proteins out of the cells. In a final step 13, the DNA-bound proteins in the intact target cells are detected. Detection 13 can include quantification of the DNA-bound proteins.

[0102] Fig. 2 schematically shows the sequence of an exemplary method 100 for fixing DNA-binding proteins in single cells of a cell mixture 1. The method 100 is carried out at a temperature of 4 °C, for example on ice, in order to keep possible enzyme activities low.

[0103] In Figure 2, in a first step a, a single cell / target cell 2 is shown, which has been highlighted as an example from a cell mixture 1. The target cell 2 has a cell nucleus 2.1 with a nuclear membrane 2.2 and the DNA strands 2.4 located in the cell nucleus 2.1, usually in the form of a double helix. The target cell 2 is surrounded by a cell membrane 2.3. For the sake of clarity, the described components of the target cell 2 are only labeled in the first step a. In the later steps bf, the components are only highlighted individually if they are again important for understanding the exemplary method 100.

[0104] Also visible in Figure 2 and individually labeled in the first step a are membrane proteins 5.1, DNA-binding proteins 5.2, and cofactors 5.3 (shown dotted in Figure 2 in steps ad). Membrane proteins 5.1 are referred to herein as membrane-bound proteins, i.e., proteins associated with the cell membrane 2.3 of the target cell 2, for example, receptors, transporters, and / or channels. DNA-binding proteins 5.2 are capable of binding to the DNA strand 2.4 and, as such, are freely mobile in the cytoplasm, as well as capable of passing through the nuclear membrane 2.2. The term cofactor 5.3 refers here to proteins that interact directly with DNA-binding proteins. In contrast to chemical methods, these proteins are not fixed to the DNA-binding protein or DNA-binding protein 5.2 or the DNA by UV laser radiation and thus washed out in a later process step. In the first step a of Figure 2, an example is a cofactor 5.3, which interacts with a DNA-bound DNA-binding protein 5.2. For the sake of clarity, membrane proteins 5.1, DNA-binding proteins 5.2, and cofactors 5.3 are also shown in detail in Figure 2 only in the first step a and are highlighted individually in steps b if they are again relevant for understanding the exemplary method 100.

[0105] After providing the target cell 2 contained in the cell mixture 1, the cell mixture 1 is irradiated with UV rays 3. The UV rays 3 are in the form of high-intensity laser beams with a wavelength in the range of 240 nm to 280 nm, preferably in the range of 250 nm to 270 nm, and an energy density of at least 10 mJ / cm 2 , preferably at least 20 mJ / cm 2 , particularly preferably at least 40 mJ / cm 2 per pulse. The maximum energy density is 1000 mJ / cm 2 .

[0106] Irradiation with UV rays 3 causes DNA-binding proteins 5.2, which were already bound to DNA strand 2.4 at the time of irradiation, to form a covalent bond with the DNA. These proteins are shown hatched in step b as proteins 5.4 covalently bound to the DNA. At the same time, UV rays 3 cause both the cell membrane 2.3 and the nuclear membrane 2.2 to become porous, thereby increasing the permeability of the cell membrane 2.3 and the nuclear membrane 2.2. The increase in permeability is shown in step c, with steps b and c usually occurring simultaneously.

[0107] The target cells 2 are then incubated in a washing liquid 200. In the exemplary embodiment, the following washing liquid 200 is selected: 0.5 M NaCl, 0.02 M HEPES, 2 wt.% BSA, protease inhibitor (e.g., 10 pg / mL aprotinin, 10 pg / mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF)), 0.033 wt.% digitonin, 0.1 wt.% octoxinol 9, although different compositions can of course also be used. The permeability of the cell membrane 2.3 and the nuclear membrane 2.2 can be further increased by optionally containing substances in the washing liquid. In particular, by incubating the target cells 2 in the washing liquid, both cofactors 5.3 and DNA-binding proteins 5.2 that are not covalently bound to the DNA are washed out of the cell nucleus 2.1 and the target cell 2. In Figure 2, the washing out in step d is graphically illustrated by arrows. Cofactors 5.3 that are bound to proteins 5.2 covalently bound to the DNA are also washed out.4 are washed out. In the target cell 2, only the membrane proteins 5.1 and the proteins 5.4 covalently bound to the DNA remain.

[0108] In a next step of the exemplary method 100, shown as step e in Figure 2, detection reagents 4 are added to the target cells 2. The detection reagents 4 are present, for example, as fluorescently labeled antibodies. Different fluorescently labeled antibodies can be used (even simultaneously) to detect various proteins 5.4 covalently bound to the DNA. The remaining membrane proteins 5.1 can also be detected using detection reagents 4, such as fluorescently labeled antibodies.

[0109] After the addition of the detection reagents 4, a final step (shown as step f in Figure 2) involves a high-throughput individual analysis, for example by flow cytometry and / or microscopy.

[0110] In the example, 2.5 million cells in 200 μl of PBS are irradiated for 10 seconds with an Nd:YAG laser. The laser delivers 30 mJ (variably adjustable) per pulse at a pulse frequency of 10 Hz (fixed), a pulse length of 5 ns (fixed), and a beam diameter of 6 mm = 0.283 cm. 2 (fixed). Optical attachments such as lenses are not used in this exemplary embodiment. The use of a prism to deflect the beam may be provided.

[0111] Figure 3 shows an exemplary experimental setup for carrying out the proposed method 100, for example the method 100 described above with reference to Figure 2. A cell mixture 1 with target cells 2 is located in a flat-bottomed reaction vessel and is held in suspension by means of a magnetic stirrer 9. The target cells 2 are irradiated for a limited time, for example, for a duration of 10 seconds, using a high-energy laser 6 with a wavelength of 266 nm + / - 10 nm and a pulse frequency of 5 ns. A neodymium-doped yttrium aluminum garnet laser (Nd:YAG laser), for example, can be used as the laser. In the illustrated embodiment, the UV rays 3 first pass through a shutter 7 and then a prism 8, via which they are directed onto the cell mixture 1.By arranging various additional optics, the photon density in the beam can be increased, allowing the device to be significantly reduced in size. Additional optics can, for example, be lenses positioned in the beam path between the laser and the sample, i.e., the target cells 2. An arrangement directly in front of the sample, i.e., in front of the cells to be irradiated, is preferable to avoid damage to other optics. An increase in the power density of the laser beam, e.g., by narrowing or focusing using optics, is thus only performed immediately in front of the sample, thus preventing high-intensity UV radiation from hitting the optics and the associated wear.

[0112] Figure 4 shows the graphical representation of the results of flow cytometry after performing the exemplary method 100.

[0113] The transcription factor STAT 1 is involved in the upregulation of genes induced by an interferon-ß (IFN-ß or IFNb) signal. In response to IFN-ß stimulation, STAT 1 forms a heterodimer with STAT 2, which translocates into the nucleus and binds to ISRE (interferon-stimulated response element) promoter elements. Binding of the promoter elements leads to increased expression of ISG (interferon-stimulated genes).

[0114] Target cells 2 were treated with 100 ng / ml IFNβ for 20 minutes to induce STAT1 DNA binding. Protein was then fixed by UV irradiation according to the exemplary procedure, and STAT1 protein was stained using a specific allophycocyanin (APC)-coupled STAT1 antibody. UV irradiation of both untreated and IFNβ-treated cells was performed at increasing doses. Measurement was performed using a flow cytometer.

[0115] Fig. 4a shows the results for irradiation with 0 mW (effectively, no irradiation of the target cells). The proportion of STAT1-positive cells was 27.3%, meaning that STAT1 proteins were detected in 27.3% of the cells.

[0116] Fig. 4b shows the results obtained with 100 mW irradiation. The proportion of STAT1-positive cells was 51.6%, meaning STAT1 proteins were detected in 51.6% of the cells. Fig. 4c shows the results obtained with 400 mW irradiation. The proportion of STAT1-positive cells was 89.3%, meaning STAT1 proteins were detected in 89.3% of the cells.

[0117] Example 1 : Significance of the proposed method for the detection of acute promyelocytic leukemia

[0118] Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia. It is characterized by a block in the differentiation of myeloid precursor cells into mature cells of the myeloid lineage and increased proliferation. Approximately 97% of APL patients carry the chromosomal translocation t(15;17), which leads to the production of the promyelocytic leukemia protein (PML)-retinoic acid receptor alpha (RARA) fusion protein (PML-RARA), which is responsible for malignancy. Due to the aggressive disease course and early life-threatening hemorrhagic and thrombotic complications, it is considered a medical emergency.

[0119] Complete blood count, coagulation parameters, peripheral blood smear, bone marrow biopsy, and immunophenotyping are recommended for all patients admitted to the hospital with suspected leukemia. Although the results of these tests may be suggestive of APL, genetic testing is mandatory, as the presence of the PML-RARA fusion determines response to ATRA / ATO treatment [chemotherapy-free treatment with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO)].

[0120] Therefore, access to early treatment and confirmation of the genetic diagnosis are crucial and life-saving for patients with APL. Due to the risks posed by the disease in its early stages and the high efficacy of treatment, testing for APL is recommended for all patients diagnosed with acute myeloid leukemia.

[0121] Currently, several strategies are actively used in the clinic for the identification of PML-RARA rearrangements: conventional karyotyping, fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (RT-PCR), or monoclonal anti-PML antibodies. However, all of these methods are time-consuming and not easily automated.

[0122] Conventional flow cytometry can be used to stain for PML in APL cells, but due to non-selective cross-linking with formaldehyde, the PML-RARA fusion protein is fixed throughout the cell. Therefore, it cannot distinguish between healthy PML-containing cells and PML-RARA-mutated APL cells.

[0123] In the proposed method, the proteins are cross-linked exclusively to DNA. RARA, but not PML, can bind to DNA. Therefore, UV laser irradiation can only fix the PML-RARA fusion, but not PML in healthy cells. After washing out the non-cross-linked PML, consecutive staining reveals the PML-RARA-containing APL cells, enabling early, specific treatment of APL patients with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO).

[0124] The cells can be washed out with an aqueous wash solution, for example, a wash buffer consisting of 0.5 M NaCl, 0.02 M HEPES, 2 wt% BSA, protease inhibitor, 0.033 wt% digitonin, and RNaseA 50 μg / ml. Optionally, 0.1 wt% octoxinol 9 can be added as a non-ionic detergent. The PML-RARA fusion protein can be detected using a dye-labeled antibody directed against PML.

[0125] Example 2: Monitoring of cancer treatment with DNA demethylation agents by the proposed method

[0126] DNA methylation is one of the well-defined mechanisms in the epigenetic control of gene expression. A group of DNA-binding enzymes, the DNA methyltransferases (DNMTs), catalyze DNA methylation by covalently attaching a methyl group to the 5-carbon atom of the cytosine ring, leading to the formation of 5-methylcytosine (5mC) at CpG regions. DNA methylation is thought to block gene expression either by directly interfering with the binding of transcription factors (TFs) to CpG regions or indirectly through the proteins that recognize methylated CpGs.

[0127] Non-mutational epigenetic reprogramming is one of the emerging hallmarks and is defined as one of the defining features in cancer. Aberrant hypermethylation of tumor suppressor genes is highly involved in the development and progression of cancer, making this mechanism an attractive target for therapeutic development. The nucleoside analog DNMT inhibitors 5-azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine) are extensively studied in the context of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) and are included in current treatment guidelines. These drugs incorporate into DNA and bind to DNMTs, leading to DNMT degradation. Azacitidine targets DNMT 1, while decitabine can target both DNMT 1 and 3A.After DNMT degradation, DNA methylation cannot be maintained, and CpGs lose their methylation after cell replication, leading to alterations in TF binding and activation of previously silenced transcriptional programs.

[0128] The proposed method can be used to actively monitor the demethylation reaction and binding dynamics of transcription factors after treatment with demethylating agents. This involves detecting the direct binding of transcription factors to DNA. Conventional flow cytometry applications can easily detect transcription factors and intracellular proteins, but these protocols rely on fixation of cells with formaldehyde. Formaldehyde creates non-selective cross-links between proteins and protein nucleic acids in the cell, making the detection of direct protein-nucleic acid interactions impossible. The method of the invention uses photochemical cross-linking with high-intensity UV laser radiation instead of formaldehyde cross-linking, which only creates cross-links between protein-nucleic acid molecules that are in direct contact.

[0129] After photochemical UV cross-linking, the cells are permeabilized and the non-cross-linked molecules are washed out. Following this, the transcription factor sets and surface antigens of interest are labeled with fluorochrome-conjugated antibodies. For example, DNMT3A and / or MeCP2 antibodies can be used. The fluorescence signals are measured and analyzed using conventional flow cytometry.

[0130] DNMT1 is the most abundant DNMT, primarily responsible for maintaining methylation, and can be targeted by DNA demethylating agents. NRF1 is also a unique methylation-sensitive transcription factor that can only bind to unmethylated DNA. It has been shown to have more binding sites in cells with low methylation status and upon global removal of DNA methylation. Using the method of the invention, downregulation of DNMT1 and increased binding of NRF1 to DNA can be detected in leukemia cells treated with demethylating drugs.

[0131] Fig. 5 shows a device with microfluidics 19, which can also be used to carry out the method 100 explained above. The device has a first reservoir 14 in which the target cells 2 to be treated are present as a cell suspension. In addition, the device has a second reservoir 15 with cells with fixed DNA-bound proteins. Via an inlet tube 16, the target cells 2 to be treated in the cell suspension pass from the first reservoir 14 into the channel 20, which serves to isolate the cells and is dimensioned accordingly. DNA-binding proteins of the target cells 2 are fixed in the channel 20 before the target cells 2 are discharged via the outlet tube 17 into the second reservoir 15. The corresponding flow direction is indicated in Fig. 5 by an arrow 21.

[0132] For treating the target cells 2, a quartz window 18, i.e., a window made of UV-permeable quartz glass, is provided in channel 20. If a target cell 2 is located in the area of ​​the quartz window 18, it can be treated by UV rays 3 emitted by a laser source 6 and passing through a shutter 7 and a prism 8 to the quartz window 18, in such a way that the DNA-binding proteins contained in the target cell 2 are fixed.

[0133] Subsequently, the treated target cells 2 in reservoir 15 can be exposed to the washing liquid 200 to wash out unfixed cell components, as already explained. Subsequently, the fixed DNA-bound proteins can be detected, for which a protein stain can be used.

[0134] Some aspects of the present invention can be summarized as follows:

[0135] The proposed method allows the individual cells of the cell mixtures to be preserved and not lysed. Detection of DNA-protein binding takes place in the "externally intact" cell. This represents a fundamental difference from all previously published methods.

[0136] The following factors can contribute to this:

[0137] 1. The cells can be specifically irradiated to ensure efficient crosslinking of proteins and DNA while maintaining cell integrity and preventing damage to the epitopes of the proteins to be detected. a. Irradiation can be performed in cold liquid to limit damage to the cell membrane and the proteins to be detected; b. Irradiation is performed with an upper energy density of 1000 mJ / cm 2to ensure cell integrity and prevent damage to the protein epitopes to be detected by antibodies; c. Irradiation can be performed with long laser pulses (ps or ns): Efficient crosslinking occurs in intact cells preferably using long pulses. Femtosecond pulses are usually inefficient in intact cells. d. Optimal crosslinking occurs through single irradiation of a cell, since multiple irradiations can destroy generated crosslinks. i. Irradiation of the cells can be performed in suspension in a tube with a stirrer: Cell concentration and irradiation time (e.g. 10 million cells in 200 ml, 5 cycles x 10 Hz = 50 pulses at 500 rpm stirring) can be precisely adjusted so that the majority of the cells are hit by the laser only once. ii. Alternative microfluidics: The cell flow can be adjusted so that each cell is hit by exactly one pulse. 2.Using a specific procedure, pores can be generated in the cells that are large enough to remove non-crosslinked protein complexes while leaving the cells intact; non-covalent DNA-protein and protein-protein bonds can be broken, and proteins non-covalently bound to the DNA can be mechanically washed out. a. Pore generation, e.g., using digitonin, etc. b. Washing out proteins non-covalently bound to DNA in the intact cell. This can be influenced by the choice of buffer and the manner in which the mechanical washing procedure is carried out.

[0138] Various aspects of the invention relate to

[0139] 1. A method for detecting and quantifying DNA-bound proteins in single cells of a cell mixture (1), comprising the steps of a) providing target cells (2), b) irradiating the target cells (2) with UV rays (3) of a wavelength in the range from 240 nm to 280 nm, preferably in the range from 250 nm to 270 nm and an energy density of at least 5 mJ / cm 2 , preferably at least 15 mJ / cm 2 , particularly preferably at least 30 mJ / cm2, and c) incubating the target cells (2) in a washing buffer.

[0140] 2. Method according to aspect 1, wherein the irradiation of the target cells (2) is carried out with pulsed UV laser radiation.

[0141] 3. Method according to aspect 1 or 2, wherein a UV laser in combination with at least one light-optical and / or electron-optical system is used to irradiate the target cells (2).

[0142] 4. The method according to any one of aspects 1 to 3, wherein the wash buffer contains at least one cholesterol-dissolving and / or pore-inducing substance, preferably polyoxyethylene(20) sorbitan monolaurate and / or a saponin, preferably digitonin, and / or leucoperm. 5. The method according to any one of aspects 1 to 4, wherein, after incubating the target cells (2) in a wash buffer, at least one detection reagent (4), preferably a fluorescently labeled antibody, is added to the irradiated target cells (2), wherein the detection reagent (4) interacts with proteins (5.4) covalently bound to the DNA.

[0143] 6. Method according to aspect 5, wherein after the addition of at least one detection reagent (4), the detection of an interaction between the detection reagent (4) and the proteins (5.4) covalently bound to the DNA takes place, preferably by means of flow cytometry and / or microscopy.

[0144] 7. Method according to one of aspects 1 to 6, wherein at least individual method steps are carried out in a multiwell plate or in or on a microfluidic component.

[0145] As used herein, the term "and / or," when used in a series of two or more elements, means that any of the listed elements may be used alone, or any combination of two or more of the listed elements may be used. For example, if a composition is described containing components A, B, and / or C, the composition may contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0146] It is understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present invention. Therefore, the foregoing description is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims.

[0147] List of reference symbols

[0148] Cell mixture

[0149] Target cell

[0150] cell nucleus

[0151] nuclear membrane

[0152] cell membrane

[0153] DNA strand

[0154] UV rays

[0155] Detection reagent

[0156] membrane protein

[0157] DNA binding protein

[0158] Cofactor

[0159] Protein covalently bound to DNA

[0160] Laser source

[0161] Shutter

[0162] prism

[0163] Magnetic stirrer

[0164] Providing target cells

[0165] Irradiation of the target cells

[0166] Incubating the target cells

[0167] Detect first reservoir second reservoir

[0168] Inlet hose

[0169] drain hose

[0170] Quartz window

[0171] Microfluidics

[0172] channel

[0173] Flow direction

[0174] Proceedings

[0175] Washing liquid

Claims

Patent claims 1. Method (100) for fixing DNA-binding proteins (5.2) in cells, the method (100) comprising the steps of: a) providing (10) target cells (2), b) irradiating (11) the target cells (2) with UV rays (3) of a wavelength in the range of 240 nm to 280 nm and an energy density in the range of 0.01 mJ / cm 2 up to 1000 mJ / cm 2 , c) incubating (12) the target cells (2) in a washing liquid (200) and washing out non-DNA-bound proteins from the target cells (2).

2. Method (100) according to claim 1, comprising: d) detecting (13) covalently DNA-bound proteins (5.4) in the target cells (2).

3. Method (100) according to one of the preceding claims, wherein the irradiation of the target cells (2) is carried out with pulsed UV laser radiation.

4. Method (100) according to one of the preceding claims, wherein when irradiating the target cells (2), the majority of the target cells (2) are irradiated exactly once.

5. Method (100) according to one of the preceding claims, wherein a UV laser in combination with at least one light-optical and / or electron-optical system is used to irradiate the target cells (2).

6. Method (100) according to one of the preceding claims, wherein the washing liquid (200) contains at least one cholesterol-dissolving and / or pore-inducing substance.

7. The method (100) according to claim 6, wherein the cholesterol-dissolving and / or pore-inducing substance is polyoxyethylene (20) sorbitan monolaurate and / or a saponin.

8. The method (100) according to any one of the preceding claims, wherein the washing liquid (200) contains at least one non-ionic detergent.

9. The method (100) of claim 8, wherein the nonionic detergent is octoxinol 9.

10. The method (100) according to any one of the preceding claims, wherein the washing liquid (200) contains at least one salt.

11. The method (100) of claim 10, wherein the salt is selected from a group comprising NaCl, LiCl, KCl, MgCh and CaCh.

12. The method (100) according to claim 10 or 11, wherein the concentration of the salt is in a range of 300 mM to 700 mM, preferably in a range of 450 mM to 550 mM.

13. The method (100) according to claim 10 or 11, wherein the concentration of the salt is in a range of 50 mM to 100 mM, preferably in a range of 75 mM to 100 mM.

14. Method (100) according to one of the preceding claims, wherein the incubation (12) of the target cells (2) in the washing liquid (200) takes place with continuous movement of the cells (2).

15. The method (100) according to any one of the preceding claims, wherein the incubation (12) of the target cells (2) in the washing liquid (200) takes place for a period of 5 minutes to 60 minutes.

16. Method (100) according to one of the preceding claims, wherein the irradiation (11) of the target cells (2) is carried out with UV rays (3) of a wavelength in the range from 250 nm to 270 nm.

17. Method (100) according to one of the preceding claims, wherein the irradiation (11) of the target cells (2) with UV rays (3) with an energy density in the range of 5 mJ / cm 2 up to 1000 mJ / cm 2 , preferably 40 mJ / cm 2 up to 250 mJ / cm 2 , takes place.

18. Method (100) according to one of the preceding claims, wherein at least individual method steps (10, 11, 12, 13) are carried out in a multi-well plate or in a microfluidic component or on a microfluidic component.

19. Method (100) according to one of the preceding claims, wherein the irradiation (11) of the target cells (2) takes place at a maximum temperature of 10 °C.

20. The method (100) according to any one of claims 2 to 19, wherein the detection (13) of the covalently DNA-bound proteins comprises adding at least one detection reagent (4) to the irradiated target cells (2), wherein the detection reagent (4) interacts with the covalently DNA-bound proteins (5.4).

21. The method (100) according to claim 20, wherein the detection reagent (4) comprises at least one fluorescently labeled antibody.

22. Method (100) according to claim 20 or 21, wherein after the addition of the at least one detection reagent (4) a detection (13) of an interaction between the detection reagent (4) and the covalently DNA-bound proteins (5.4) takes place.

23. The method (100) according to claim 22, wherein the detection (13) of the interaction is carried out by means of flow cytometry and / or microscopy.

24. Washing liquid (200) comprising: a) at least one cholesterol-dissolving and / or pore-inducing substance, b) at least one non-ionic detergent, and c) at least one salt.

25. Washing liquid (200) according to claim 24, wherein the cholesterol-dissolving and / or pore-inducing substance is polyoxyethylene (20) sorbitan monolaurate and / or a saponin.

26. Washing liquid (200) according to claim 24 or 25, wherein the non-ionic detergent is Octoxinol 9.

27. Washing liquid (200) according to one of claims 24 to 26, wherein the salt is selected from a group comprising NaCl, LiCl, KCl, MgCh and CaCh.

28. Washing liquid (200) according to one of claims 24 to 27, wherein the concentration of the salt is in a range of 300 mM to 700 mM, preferably in a range of 450 mM to 550 mM.

29. Washing liquid (200) according to one of claims 24 to 27, wherein the concentration of the salt is in a range of 50 mM to 100 mM, preferably in a range of 75 mM to 100 mM.

30. Use of a washing liquid (200) according to any one of claims 24 to 29 for incubating cells.

31. Use of a washing liquid (200) according to one of claims 24 to 29 for permeabilizing cell membranes and / or cell nuclear membranes.

32. Use of a washing liquid (200) according to any one of claims 24 to 29 for washing out non-DNA-bound proteins from cells.

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