Washing device

Abstract

Washing device (1), comprising: - a laboratory container holder (2) comprising M means for securing laboratory containers (3), with M>1, said means for securing (3) being arranged along at least one axis (x); - a stirring means (5) adapted to be reversibly attached to said laboratory container holder (2) and adapted to induce rotations about an axis of rotation (y) parallel to said at least one axis (x) of said laboratory container holder (2) when the latter is attached to said stirring means (5); - a base (6) for the laboratory container holder (2) comprising at least one magnet, said base (6) being adapted to be reversibly attached to said laboratory container holder (2). Figure to be published with the abbreviation: Fig. 1

Description

Title of the invention: Washing device

[0001] The use of magnetic particles, and in particular magnetic beads, in purification processes is widespread. This often involves, but is not limited to, the purification of cells or biological macromolecules such as isolated proteins, antibodies, and nucleic acids.

[0002] Magnetic particles, in particular magnetic beads, are used to purify these cells and / or these biological macromolecules.

[0003] The element to be purified attaches itself to the magnetic particles, which allows it to be separated from the surrounding medium.

[0004] The protocols enabling these purifications involve, among other things, several successive washing steps of these magnetic particles.

[0005] Typically, these washes are carried out according to the following simplified protocol:

[0006] - the magnetic particles are arranged in test tubes with presence of a washing solution, these tubes being located on a rack;

[0007] - the test tubes are moved one by one from the rack to a stirrer;

[0008] - the test tubes are shaken in order to resuspend the magnetic beads in the washing solution and thus wash them;

[0009] - after stopping the stirring, the test tubes are removed one by one from the stirrer and placed on a magnetic rack. Thanks to the action of the nearby magnetic field, the magnetic particles are pressed against the walls of the test tubes;

[0010] - finally, the soiled washing solution is removed, for example by means of using a pipette, and changed with a clean washing solution.

[0011] These steps are repeated a number of times until the magnetic particles are properly washed, according to the recommendations depending on their intended use. More than ten, or even more than twenty, washes may be necessary, depending on the protocols applied.

[0012] This washing protocol, although functional, has a number of drawbacks.

[0013] In particular, it requires a large number of human interventions and repetitive actions, which are likely to be sources of errors, in particular sample inversions, and of inconvenience for laboratory staff (hand fatigue, etc.).

[0014] Next, the repeatability of the shaking, that is to say the extent to which the homogenization was the same from one test tube to another, is imperfect.

[0015] For example, when 24 test tubes are to be treated simultaneously and 5 successive washes are recommended, this is very tedious and the test tubes are handled hundreds of times (24 multiplied by 5 multiplied by 2, therefore 240 manipulations), which takes time and can lead to sample inversions, fatigue of laboratory staff, etc.

[0016] It should be noted that a handling error, for example a sample reversal, will be very difficult, or even impossible, to detect and may invalidate the entire manipulation.

[0017] In addition, an almost infinite amount of time may be spent trying to find the source of the error, without systematically identifying it.

[0018] In the prior art, certain steps have been taken with regard to laboratory equipment.

[0019] In application CN107402154, a test tube holder permanently fixed to a rotation axis is described.

[0020] In application CN207976333, a one-block device is described that has the possibility of "back-and-forth" agitation between two electromagnets.

[0021] These solutions are not satisfactory.

[0022] For the purposes of this application, "laboratory container" means any laboratory equipment capable of containing, in particular, solutions or suspensions. This may include, for example, test tubes that can be closed with a suitable stopper or any other means of closure.

[0023] In the context of this application, the interval described as "between A and B" includes the bounds "A" and "B".

[0024] Surprisingly, it was highlighted by the applicant that a laboratory magnetic particle washing device 1, which improves the washing process and has a large number of advantages, could be made.

[0025] The invention therefore relates first of all to a washing device 1, comprising:

[0026] - a laboratory container holder 2 comprising M fastening means 3 of laboratory containers, with M>1, said fixing means 3 being arranged along at least one axis (x);

[0027] - an agitation means 5 capable of being reversibly linked to said container holder laboratory 2 and capable of inducing rotations about an axis of rotation (y) parallel to at least one axis (x) of said laboratory container holder 2 when the latter is linked to said stirring means 5;

[0028] - a laboratory container base 6 comprising at least one magnet, said base 6 being capable of being reversibly linked to said laboratory container holder 2.

[0029] In one embodiment, said washing device 1 is a laboratory magnetic particle washing device, for example magnetic balls.

[0030] In one embodiment, the laboratory magnetic particles have a larger dimension between 10 nanometers and 1 millimeter.

[0031] In one embodiment, the laboratory magnetic particles have a larger dimension of between 20 and 100 nanometers.

[0032] In one embodiment, the laboratory magnetic balls have a diameter between 20 and 100 nanometers.

[0033] In one embodiment, the laboratory magnetic particles have a larger dimension between 100 nanometers and 1 millimeter.

[0034] In one embodiment, the laboratory magnetic particles have a larger dimension between 200 nanometers and 1 millimeter.

[0035] In one embodiment, the laboratory magnetic particles have a larger dimension between 10 nanometers and 100 micrometers.

[0036] In one embodiment, the laboratory magnetic particles have a larger dimension of between 1 and 5 micrometers.

[0037] In one embodiment, the laboratory magnetic particles have a larger dimension of about 2.8 micrometers.

[0038] In one embodiment, the laboratory magnetic balls have a diameter between 10 nanometers and 100 micrometers.

[0039] In one embodiment, the laboratory magnetic balls have a diameter between 1 and 5 micrometers.

[0040] In one embodiment, the magnetic balls have a diameter between 10 nanometers and 1 millimeter.

[0041] In one embodiment, the magnetic balls have a diameter between 100 nanometers and 1 millimeter.

[0042] In one embodiment, the magnetic balls have a diameter between 200 nanometers and 1 millimeter.

[0043] In one embodiment, all non-electrical parts of said washing device 1 are made of a material that is not susceptible to corrosion.

[0044] In one embodiment, all non-electrical parts of said washing device 1 are made of a plastic material and / or resin.

[0045] In one embodiment, all non-electrical parts of said washing device 1 are made of a plastic material.

[0046] In one embodiment, all non-electrical parts of said washing device 1 are made of resin.

[0047] In one embodiment, all non-electrical parts of said device 1 are made of a material selected from the group consisting of polylactic acid (PLA) and glycolized polyester (PETG).

[0048] In one embodiment, all non-electrical and non-magnetic parts of said washing device 1 are three-dimensionally printed, and are made of a material selected from the group consisting of polylactic acid (PLA) and glycolized polyester (PETG).

[0049] Three-dimensional printing (3D printing) has the advantage of being very simple to implement. Furthermore, it also allows the insertion of magnets into the base 6 of the laboratory container holder 2, thus enabling the production of a single part. In addition, certain parts of the stirring means 5 are hollow with cavities allowing the passage of electrical cables and the placement of electrical components (motor, etc.), which 3D printing makes possible. Finally, this limits non-watertight joints, and therefore contributes to the sealing / tropicalization of the stirring means 5.

[0050] In one embodiment, said base 6 of laboratory container holder 2 comprising at least one magnet is capable of being reversibly linked to said laboratory container holder 2 and of collecting laboratory magnetic particles within said laboratory containers.

[0051] In one embodiment, said laboratory container holder 2 comprises a carrier plate 7 and a detachable stabilizing base 8 for said carrier plate. Thus, the magnetic base 6 can, for example, be inserted between the carrier plate 7 and the detachable stabilizing base 8. Furthermore, in this embodiment, the carrier plate 7 can be changed while retaining the same detachable stabilizing base. Thus, when specific laboratory containers different from those previously used need to be processed, the user simply needs to change the carrier plate 7.

[0052] In one embodiment, said laboratory container holder 2 comprises a plurality of carrier plates 7 and a detachable stabilizing base 8 from said carrier plates 7. In this embodiment, each carrier plate 7 can be used separately with the stabilizing base 8, in particular to carry out agitation by means of the stirrer 5.

[0053] In one embodiment, said laboratory container holder 2 comprises a plurality of carrier plates 7 and a detachable stabilizing base 8 of said carrier plates 7, said carrier plates being adapted to different laboratory containers.

[0054] In one embodiment, said laboratory container holder 2 comprises a plurality of carrier plates 7 and a stabilizing base 8 detachable from said carrier plates 7, said carrier plates comprising fastening means 3 different in number of fastening means and / or in dimensions of fastening means.

[0055] In one embodiment, said laboratory container holder 2 comprises a plurality of carrier plates 7 and a detachable stabilizing base 8 for said carrier plates 7, and said carrier plates 7 are each a different color. Thus, they will be very easily distinguishable and will save the user time.

[0056] In one embodiment, said device 1 comprises between 2 and 10 different carrier plates 7.

[0057] In one embodiment, said device 1 comprises between 2 and 10 different carrier plates 7 having different colours.

[0058] In one embodiment, said support plate includes several feet 4, said fixing means 3 are holes with flats, and said detachable stabilizing base 8 includes fixing means for said stirring means 5.

[0059] In one embodiment, said laboratory container holder 2 includes stabilization means, preferably feet 4. Thus, according to the user's wishes, the support plate 7 can serve as a carrier even in the absence of a stabilizing base 8, because it is stabilized by the feet 4.

[0060] In one embodiment, the laboratory container holder 2 is constituted by said carrier plate 7.

[0061] In one embodiment, said laboratory container holder 2 includes stabilization means enabling it to hold in a position enabling it to accommodate the laboratory containers vertically to prevent their contents from tipping over.

[0062] In a particularly preferred embodiment, these stabilization means are 4 feet. This is particularly advantageous, as it allows for complete stabilization.

[0063] Thus, whether or not the laboratory container holder 2 is equipped with laboratory containers, it will remain stable, and all the more stable the higher the number of feet 4 is.

[0064] In one embodiment, said laboratory container holder 2 comprises a number of feet 4 between 2 and 10, preferably between 3 and 7, and preferably 5.

[0065] In a preferred embodiment, said laboratory container holder 2 comprises 3 feet 4.

[0066] In a preferred embodiment, said laboratory container holder 2 comprises 5 feet 4.

[0067] It is useful to provide a large number of feet 4, however it is also necessary to provide spaces between the feet 4 in which the magnets can to be inserted. Thus, it was shown that a number of feet 4 between 2 and 10 was optimal.

[0068] In one embodiment, M is between 2 and 48, preferably between 6 and 36, preferably between 16 and 24.

[0069] Here again, the aim is to have as many means of fixing 3 as possible, while maintaining an overall dimension of the washing device 1 compatible with easy handling.

[0070] In one embodiment, said laboratory container holder 2 includes fastening means 3 of different kinds, and more specifically which allow different laboratory containers to be fixed.

[0071] The various laboratory containers may, in particular, have different volumetric capacities. Thus, by means of the washing device 1, it will be possible to wash magnetic particles, including within tubes of different capacities, within the same laboratory procedure.

[0072] For example, the laboratory container holder 2 may include 24 fastening means 3 (M=24), of which 4 fastening means 3 are intended for fastening 4 laboratory containers with a capacity of 15 millilitres, and 20 fastening means 3 are intended for fastening 20 laboratory containers with a capacity of 1.5 millilitres.

[0073] In one embodiment, at least one fastening means 3 is adapted for fastening a laboratory container chosen from the group of laboratory containers with capacities of 0.5 millilitres, 1.5 millilitres, 2 millilitres, 5 millilitres, 15 millilitres, or 50 millilitres.

[0074] In one embodiment, at least two fastening means 3 are adapted for fastening a laboratory container chosen from the group of laboratory containers with capacities of 0.5 millilitres, 1.5 millilitres, 2 millilitres, 5 millilitres, 15 millilitres, or 50 millilitres.

[0075] In one embodiment, at least five fastening means 3 are adapted for fastening a laboratory container chosen from the group of laboratory containers with capacities of 0.5 millilitres, 1.5 millilitres, 2 millilitres, 5 millilitres, 15 millilitres, or 50 millilitres.

[0076] In one embodiment, said magnet is capable of gathering said laboratory magnetic particles within said laboratory containers.

[0077] In one embodiment, said magnet is capable of gathering said laboratory magnetic particles within said laboratory containers, said gathering taking place at least 1 millimeter above the bottom of said laboratory container. This embodiment is preferred because if a pipette is used to remove the contaminated medium, then removal of the contaminated medium without removing magnetic particles will be facilitated.

[0078] In one embodiment, at least one fastening means 3, and preferably all of the fastening means 3, are holes capable of mechanically retaining laboratory containers.

[0079] Thanks to 3D printing, it is possible to accommodate any container by creating a custom-made hole. For example, for certain experiments involving the use of highly specialized new laboratory containers, it will be possible to produce a suitable laboratory container holder 2 using 3D printing. This is particularly advantageous in a preferred embodiment, in which the laboratory container holder 2 includes the support plate 7 and the stabilizing base 8. Indeed, in this latter case, it will suffice to 3D print a new support plate 7 to have a complete device adapted to these highly specialized new laboratory containers.

[0080] Preferably, these holes have a flat part allowing better mechanical action of said hole on said laboratory containers; they are referred to in the context of this application as “holes with flats”.

[0081] In one embodiment, at least one fastening means 3, and preferably all of the fastening means 3, are holes with flats capable of mechanically retaining laboratory containers.

[0082] In one embodiment, said fastening means 3 are arranged on said axis (x). This embodiment is particularly suitable for obtaining strictly identical agitation between different laboratory containers. Indeed, all laboratory containers are subjected to exactly the same mechanical stresses during agitation.

[0083] In one embodiment, said fastening means 3 are arranged on either side of said at least one axis (x). This embodiment is particularly suitable for maximizing the number of fastening means 3 while maintaining a reduced overall size of the laboratory container holder 2.

[0084] In one embodiment, said fastening means 3 are arranged along two axes (xl) and (x2). This embodiment is particularly preferred, as it allows for the simultaneous agitation of a large number of laboratory containers.

[0085] In one embodiment, said fastening means 3 are arranged along two axes (xl) and (x2), both parallel to the axis of rotation (y).

[0086] In one embodiment, said fastening means 3 are arranged on the axes (xl) and (x2), both parallel to the axis of rotation (y).

[0087] In a preferred embodiment, said fastening means 3 are arranged along two axes (xl) and (x2), both parallel to the axis of rotation (y), and (xl) and (x2) are equidistant from said axis of rotation (y).

[0088] In a preferred embodiment, said fastening means 3 are arranged on the axes (xl) and (x2), both parallel to the axis of rotation (y), and (xl) and (x2) are equidistant from said axis of rotation (y).

[0089] In one embodiment, said stirring means 5 is a rotary stirrer.

[0090] In one embodiment, said stirring means 5 is a rotary stirrer with drum.

[0091] In one embodiment, said stirring means 5 is powered by electric current.

[0092] In one embodiment, said stirring means 5 comprises a power supply and an electric motor.

[0093] In one embodiment, said stirring means 5 includes a power supply, an electric motor, and a controller 9.

[0094] In one embodiment, said stirring means 5 includes a display screen 10. This screen can, for example, display the number of revolutions per minute as well as a countdown timer.

[0095] In one embodiment, the controller 9 allows the following parameters to be predetermined: the rotation speed, determined by the number of rotations per minute when the rotations are complete or the number of "back and forth" movements per minute when the translations are in "back and forth", as well as the duration of the agitation (in minutes).

[0096] In one embodiment, the controller 9 allows the following parameters to be determined: the rotation speed, determined by the number of rotations per minute when the rotations are complete or the number of "back and forth" movements per minute when the translations are in "back and forth", as well as the duration of the agitation (in minutes).

[0097] In one embodiment, the controller 9 allows the following parameters to be predetermined: the rotation speed, as well as the duration of the agitation.

[0098] In one embodiment, the controller 9 allows the following parameters to be determined: the rotation speed, as well as the duration of the agitation.

[0099] In a particularly preferred embodiment, said stirring means 5 is powered by electricity from a rechargeable battery. This is particularly advantageous, as it greatly improves the stirrer's mobility. For example, it can be placed inside a refrigerator if required for laboratory procedures, which would be difficult if it were powered by mains electricity. Indeed, in the prior art, the following practice has been observed: the stirrer is plugged into the mains, the stirrer is inside a refrigerator, and the power cord passes through the refrigerator seal; this is imperfect in terms of space and dangerous (risks of electrocution and short circuits, particularly due to humidity).

[0100] In one embodiment, said stirring means 5 is capable of inducing successive complete rotations. This type of rotation will be particularly suitable in general, as it induces very strong turbulence inside laboratory containers, which allows for high-quality washing.

[0101] In one embodiment, said stirring means 5 is capable of inducing successive complete rotations, and these rotations are carried out at a rate of between 10 and 100 rotations per minute and for a time of between 1 minute and 24 hours.

[0102] In one embodiment, said stirring means 5 is capable of inducing "back-and-forth" type rotations with an amplitude between 10 and 180 degrees. This type of rotation will be particularly suitable in cases where there is concern regarding the sealing of said laboratory containers.

[0103] In one embodiment, said base 6 comprises a plurality of magnets.

[0104] In one embodiment, said magnets are of grade N52.

[0105] In one embodiment, the power of said magnets is between 1 kg and 100kg.

[0106] In one embodiment, said magnets are permanent magnets.

[0107] In one embodiment, said base 6 comprises an alternation of magnets and hollowed parts compatible with the feet 4 of the laboratory container holder 2. This embodiment is particularly preferred, as it allows total stabilization of the laboratory container holder 2 within said base 6.

[0108] In one embodiment, the magnets are arranged such that the magnetic environment inside the laboratory containers is identical from one laboratory container to another. This embodiment is particularly preferred because it will allow, in particular, perfect repeatability and identity between the different laboratory containers with regard to the gathering of magnetic particles.

[0109] In the context of this application, as the person skilled in the art will immediately understand, all the terms "parallel(s)" refer in particular to one or more parallel(s) in space.

[0110] The invention also relates to a washing method using the washing device 1 according to the invention.

[0111] The embodiments described for the washing device 1 are therefore also applicable to the washing process.

[0112] The invention therefore also relates to a method for washing magnetic particles, comprising the following steps:

[0113] - step A: bringing said magnetic particles into contact with a solution of washing within several laboratory containers;

[0114] - step B: mounting the laboratory containers on a container holder laboratory 2;

[0115] - step C: mounting the laboratory container holder 2 on a stirring means 5; steps A, B and C can be carried out in any order;

[0116] - step D: agitation is started using the stirring means 5 and then stopped the agitation;

[0117] - step E: dissociation of said laboratory container holder 2 and said means 5 agitation;

[0118] - step F: mounting said laboratory container holder 2 on a suitable base 6 to the laboratory container holder 2 comprising at least one magnet capable of being reversibly linked to said laboratory container holder 2 and of gathering said magnetic laboratory particles within said laboratory containers;

[0119] - step G: removal of said washing solution.

[0120] In one embodiment, said laboratory magnetic particles are magnetic balls.

[0121] In one embodiment, the AG steps are repeated between 2 and 100 times.

[0122] In one embodiment, the AG steps are repeated between 2 and 50 times.

[0123] In one embodiment, the AG steps are repeated between 2 and 20 times. Description of the figures

[0124] [Fig.l]: Perspective view of the laboratory magnetic particle washing device 1 according to the invention, the 3 main elements being dissociated.

[0125] More specifically, it is visible both the laboratory container holder 2 comprising feet 4 and 24 laboratory container fixing means 3 which are holes with flats arranged on the axes (xl) and (x2), the stirring means 5 with a rotation axis (y) parallel to the axes (xl) and (x2) when the laboratory container holder 2 is installed on the stirring means 5, as well as the base 6 of the laboratory container holder 2 comprising twelve magnets embedded in the mass.

[0126] The base 6 also includes recesses which correspond to the feet of the laboratory container holder 2. When the laboratory container holder 2 is installed on said base 6, the feet 4 of said laboratory container holder 2 fit into the recesses of said base 6.

[0127] Two hermetically sealed laboratory containers are also visible.

[0128] [Fig.2]: Perspective view of the magnetic particle washing device 1 laboratory according to the invention, with the laboratory container holder 2 mounted on the stirring means 5.

[0129] As can be seen, when agitation is to be carried out, it is imperative that the magnetic particles be free within said laboratory containers, and Therefore, the base 6 is dissociated. For clarity, laboratory containers, such as test tubes, are not shown in this figure, although they are obviously in place during shaking. The (y), (xl), and (x2) axes are not shown for clarity.

[0130] Two hermetically sealed laboratory containers are also visible.

[0131] [Fig.3]: Perspective view of a laboratory container holder 2 according to the invention comprising 24 fixing means 3 being holes with flats.

[0132] Within this laboratory container holder 2, the holes with flats are of different sizes, and therefore adapted to different laboratory containers. It is thus possible to shake laboratory containers of different volumes at the same time.

[0133] Figure shows four types of laboratory containers of different capacities, all hermetically sealed.

[0134] [Fig. 4]: Perspective view of two different carrier plates 7. These carrier plates 7 include, in particular, different fastening means 3. These two carrier plates 7 can be adapted to the same detachable stabilizing base 8. Each carrier plate includes feet 4. The stabilizing base includes fastening means 11 to the stirring means 5 (stirring means 5 not shown). The two carrier plates 7 are different colors (not visible in the figure).

[0135] Example 1: Use of a device according to the invention in the context of an immunoprecipitation manipulation of chromatin

[0136] Immunoprecipitation of chromatin was performed on 12 samples. Chromatin was immunoprecipitated using a specific anti-P65-RELA antibody and a non-specific IgG control antibody.

[0137] The manipulation will therefore involve two times 12 samples, i.e. 24 samples.

[0138] In the context of this example, the washing device 1 used is that shown in [Fig.1],

[0139] Step I: Cross-link

[0140] 1. Prepare and label 12 tubes of 1.5 ml corresponding to the 12 samples.

[0141] 2. Wash the 12 cell boxes with 5 ml of buffered saline solution Phosphate (Phosphate Buffered Saline, hereinafter "PBS") at room temperature. Remove the PBS by vacuuming.

[0142] 3. Add 20 ml of culture medium with 1% formaldehyde per plate. Incubate for 10 minutes at room temperature on a stirrer.

[0143] 4. Add 3 ml of PBS with IM glycine to the medium to stop the reaction. Incubate for 5 minutes at room temperature on a shaker.

[0144] 5. Remove the middle and rinse twice with 5 ml of cold PBS.

[0145] 6. Aspirate the PBS and add 2 ml of cold PBS. Scrape the 12 cell boxes and the Transfer into a 5ml tube and keep on ice.

[0146] 7. Centrifuge the 12 tubes for 5 min at 1500 rpm at 4°C. Discard the supernatant by aspiration.

[0147] 8. Place the centrifuged tubes on a rack and resuspend the cell pellets in 1 ml of lysis buffer.

[0148] 9. Place the 12 tubes on a rotary shaker and incubate for 30 min at 4°C (15 rotations per minutes, hereinafter "rpm").

[0149] During this step I, the use of the device according to the invention allows a single manipulation (of the laboratory container holder) which is used from substep 8. and which is placed, during substep 9., directly on the stirring means.

[0150] As a reminder, if the classic protocol had been used, 12 more manipulations would have been required between steps 8 and 9.

[0151] Step II: Chromatin Fragmentation

[0152] 1. Retrieve the 12 tubes at 4°C and centrifuge them for 5 min at 3000 rpm at 4°C.

[0153] 2. Transfer the tubes to a rack and discard the supernatants, then resuspend the pellets in 1 ml of fragmentation buffer.

[0154] 3. Fragment the 12 samples using the Covaris.

[0155] Step III: Chromatin clarification

[0156] 1. Transfer the lysates into 12 x 1.5 ml tubes and centrifuge for 10 min at 13,000 rpm at 4°C.

[0157] 2. Transfer the supernatants into 12 1.5 ml tubes and place them on ice.

[0158] 3. Perform a phenol-chloroform extraction on the 12 tubes in order to recover purified chromatin.

[0159] 4. Perform 2% agarose gel electrophoresis on a portion of the chromatin purified to check for fragmentation.

[0160] 5. Measure the chromatin of the 12 samples to determine their concentrations.

[0161] 6. For each sample and each antibody, place 25pg of chromatin in Prepare 1.5 ml tubes and fill to 1 ml with dilution buffer. Here we have 12 samples and 2 antibodies (P65-RELA and IgG control), which gives us 24 1.5 ml tubes.

[0162] Step IV: Washing the magnetic beads

[0163] 1. Take 1.5 ml of magnetic beads and place them in a 5 ml tube.

[0164] 2. Place the 5 ml tube on a magnetic rack. Remove the supernatant.

[0165] 3. Add 4 ml of PBS and incubate on a rotary shaker for 5 min at 4°C (20 rpm).

[0166] 4. Repeat steps 2 and 3 twice (3 PBS washes in total).

[0167] 5. Place the 5 ml tube on a magnetic rack. Remove the supernatant.

[0168] 6. Add 5 ml of dilution buffer and incubate on a rotary shaker for 5 min at 4°C (20 rpm).

[0169] 7. Repeat steps 5 and 6 twice (3 dilution buffer washes in total).

[0170] 8. Place the 5 ml tube on a magnetic rack. Remove the supernatant.

[0171] 9. Add 1.5 ml of dilution buffer and incubate on wheel for 5 min at 4°C (20 rpm).

[0172] Step V: Pre-clearing

[0173] 1. Add 30 pL of washed beads (from step IV.9) into each of the 24 tubes (of step III.6). Keep the remaining marbles at 4°C.

[0174] 2. Place the 24 tubes on a rotary shaker for 30 min at 4°C (15 rpm).

[0175] 3. Place the 24 tubes on the magnetic rack. Transfer the supernatants into 1.5 ml tubes.

[0176] During this step V, with the use of the device according to the invention, the test tubes remain on the rack from the end of sub-step 1 until sub-step 3.

[0177] As a reminder, if the classic protocol had been used, it would have been necessary to perform 24 manipulations during sub-step 2. and 24 manipulations during sub-step 3..

[0178] Step VI: Immunoprecipitation

[0179] 1. Add the antibodies to the 24 tubes (12 RELA tubes and 12 IgG tubes): 5 pg of antibodies for 25 pg of chromatin.

[0180] 2. Place the 24 tubes on the rotary shaker and incubate overnight at 4°C (10-15 rpm).

[0181] 3. Place the 24 tubes on a rack.

[0182] 4. Add 30 pl of washed and stored beads at 4°C (step Vl) per tube.

[0183] 5. Place the 24 tubes on the rotary shaker and incubate for 1 to 2 hours at 4°C (10-15 rpm).

[0184] During this step VI, with the use of the device according to the invention, the test tubes remain on the rack from the end of sub-step 1 until sub-step 5.

[0185] As a reminder, if the classic protocol had been used, it would have been necessary to carry out 24 manipulations during sub-step 2, 24 manipulations during sub-step 3 and another 24 manipulations during sub-step 5.

[0186] Step VII: Washing

[0187] 1. Place the 24 tubes on the magnetic rack. Discard the supernatant.

[0188] 2. Add 1 ml wash buffer I into each tube.

[0189] 3. Place the 24 tubes on the rotary shaker and incubate for 5 min at 4°C (20 rpm).

[0190] 4. Place the 24 tubes on the magnetic rack. Discard the supernatant.

[0191] 5. Add 1 ml wash buffer II into each tube.

[0192] 6. Place the 24 tubes on the rotary shaker and incubate for 5 min at 4°C (20 rpm).

[0193] 7. Place the 24 tubes on the magnetic rack. Discard the supernatant.

[0194] 8. Add 1 ml wash buffer III into each tube.

[0195] 9. Place the 24 tubes on the rotary shaker and incubate for 5 min at 4°C (20 rpm).

[0196] 10. Place the 24 tubes on the magnetic rack. Discard the supernatant.

[0197] 11. Add 1 ml of IV wash buffer to each tube.

[0198] 12. Place the 24 tubes on the rotary shaker and incubate for 5 min at 4°C (20 rpm).

[0199] 13. Place the 24 tubes on the magnetic rack. Discard the supernatant.

[0200] 14. Add 1 ml of wash buffer V into each tube.

[0201] 15. Place the 24 tubes on the rotary shaker and incubate for 5 min at 4°C (20 rpm).

[0202] 16. Place the 24 tubes on the magnetic rack. Discard the supernatant.

[0203] 17. Add 1 ml of elution buffer to each tube.

[0204] 18. Place the 24 tubes on the rotary shaker and incubate for 5 min at 4°C (20 rpm).

[0205] 19. Place the 24 tubes on the magnetic rack. Discard the supernatant.

[0206] 20. Add 1 ml of elution buffer to each tube.

[0207] 21. Place the 24 tubes on the rotary shaker and incubate for 5 min at 4°C (20 rpm).

[0208] 22. Place the 24 tubes on the magnetic rack. Discard the supernatant.

[0209] (If the signal-to-noise ratio is too low, double all the washes above)

[0210] As a reminder, if the classic protocol had been used (without doubling the washes), it would have been necessary to carry out 360 manipulations.

[0211] Step VIII: Elution, reverse crosslink and purification

[0212] 1. Resuspend the beads in 0.5 ml of elution buffer.

[0213] 2. Add 5 pl of proteinase K (20 mg / mL) to each tube.

[0214] 3. Incubate for at least 4 hours at 65°C on the Thermomixer (1000 rpm).

[0215] 4. Add 1 volume of Phenol:Chloroform:Alcohol-Isoamyl 25:24:1 (0.5 ml).

[0216] 5. Vortex and centrifuge for 10 min at 13000 rpm at room temperature.

[0217] 6. Collect the aqueous phase.

[0218] 7. Add 1 µl of glycogen and 0.7 volume of isopropanol (0.35 ml).

[0219] 8. Vortex, centrifuge 20 min at 13000 rpm at 4°C.

[0220] 9. Remove the supernatant and add 1 ml of 70% ethanol.

[0221] 10. Vortex and centrifuge for 5 minutes at 13000 rpm at 4°C.

[0222] 11. Remove the supernatant and allow the pellets to dry with the tubes open.

[0223] 12. Resuspend the immunoprecipitated chromatin pellets in 60 µl of water without DNase (20 pl if Chip-seq).

[0224] A summary of the contribution of the use of the device according to the invention is given in Table 1.

[0225] [Tables 1] Steps Number of manipulations using the device according to the invention Number of manipulations without using the device according to the invention Cold room trips without using the device according to the invention Step I 1 12 1 Step II 0 0 1 Step III 0 0 0 Step IV 0 0 10 Step V 2 48 2 Step VI 3 72 3 Step VII (if signal / noise too weak) 15 (29) 360 (696) 15 (29) Step VII 1 0 0 0 TOTAL (if signal / noise weak) 21 (35) 492 (828) 32 (46) Table 1: Benefits of using the device according to the invention

Claims

Demands

1. Washing device (1), comprising: - a laboratory container holder (2) comprising M means for securing laboratory containers, with M>1, said means for securing (3) being arranged along at least one axis (x); - a stirring means (5) capable of being reversibly linked to said laboratory container holder (2) and capable of inducing rotations about an axis of rotation (y) parallel to said at least one axis (x) of said laboratory container holder (2) when the latter is linked to said stirring means (5), at a rate of between 10 and 100 rotations per minute and for a time of between 1 minute and 24 hours; - a base (6) for the laboratory container holder (2) comprising at least one magnet, said base (6) being capable of being reversibly linked to said laboratory container holder (2).

2. Washing device (1) according to claim 1, characterized in that said laboratory container holder 2 comprises a plurality of carrier plates 7 and a detachable stabilizing base 8 of said carrier plates 7.

3. Washing device (1) according to any one of the preceding claims, characterized in that said base 6 comprises a plurality of magnets.

4. Washing device (1) according to claim 3, characterized in that said magnets are of grade N52.

5. Washing device (1) according to any one of the preceding claims, characterized in that said laboratory container holder (2) includes stabilization means, preferably feet (4).

6. Washing device (1) according to any one of the preceding claims, characterized in that M is between 2 and 48, preferably between 6 and 36, preferably between 16 and 24.

7. Washing device (1) according to any one of the preceding claims, characterized in that at least one fastening means (3), and preferably all of the fastening means (3), are holes capable of mechanically retaining laboratory containers.

8. Washing device (1) according to any one of the preceding claims, characterized in that at least one fastening means (3), and preferably all the means of fixing (3), are holes with flats capable of mechanically retaining the laboratory containers.

9. Washing device (1) according to any one of the preceding claims, characterized in that said stirring means (5) comprises an electrical power supply, an electric motor, and a controller (9), said controller preferably allowing the following parameters to be predetermined: the rotation speed, and the duration of the stirring.

10. A method for washing magnetic particles using a washing device (1) according to any one of the preceding claims, comprising the following steps: - step A: bringing said magnetic particles and a washing solution into contact within several laboratory containers; - step B: mounting the laboratory containers on a laboratory container holder (2); - step C: mounting the laboratory container holder (2) on a stirring means (5); steps A, B and C being able to be carried out in any order; - step D: activating the stirring means (5) and then stopping the stirring; - step E: dissociating said laboratory container holder (2) and said stirring means (5);- Step F: Mounting said laboratory container holder (2) on a base (6) adapted to the laboratory container holder (2) comprising at least one magnet capable of being reversibly linked to said laboratory container holder (2) and of gathering said magnetic laboratory particles within said laboratory containers; - Step G: Removal of said washing solution.

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