Novel cell permeation agent and application

Abstract

The invention discloses a novel cell permeable agent and application, the cell permeable agent comprises tea tree oil and can further comprise rutin, the mass concentration of the tea tree oil is 0.15-0.3%, and the mass concentration of the rutin is 0.05-0.1%. The effective components of the cell permeation agent are the lipophilic tea tree oil and the rutin with strong oxidizing property, under the synergistic effect of the tea tree oil and the rutin, the cell permeation agent is strong in penetrating power, reduces cell damage, can effectively permeate adherent and suspended cells, is normal in cell morphology after permeation treatment, has no non-specific background signal, and has a good application prospect. Compared with a classical permeation method (Triton X-100, saponin and the like), the permeation agent has the advantages that the staining intensity and the positive rate of cells which are permeated by the permeation agent are equivalent or better, the cell morphology is normal, and no non-specific background signal exists.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a novel cell permeabilizing agent and its application. Background Art

[0002] Immunofluorescence (IF) is based on the principle of specific antigen-antibody binding. It utilizes a fluorescently labeled secondary antibody (such as FITC) to recognize and bind to the primary antibody-antigen complex, allowing direct localization of the target protein's distribution and expression levels in cells or tissues using fluorescence microscopy. Its applications encompass protein subcellular localization, disease marker detection, drug mechanism of action research, and clinical pathology diagnosis. Cell permeabilization agents are crucial in IF testing, disrupting the lipid bilayer structure of the cell membrane, allowing antibodies to enter the cell to detect the target antigen while potentially reducing nonspecific background signals.

[0003] Commonly used permeabilization agents include Triton X-100, NP-40, Tween-20, organic solvents, and saponin. The choice of permeabilization agent should take into account factors such as antigen localization and cell type. For example, for intracellular proteins, a strong detergent such as Triton X-100 should be used, while a mild permeabilization agent such as saponin should be used for membrane proteins. For adherent cells, use Triton, while for suspension cells, use saponin.

[0004] The selection of permeabilization agents is based on complex criteria. Permeabilization agents with strong permeabilization effects will destroy membrane proteins and cell structures, while non-destructive permeabilization agents have weak permeabilization effects. In addition, the working principle of most cell permeabilization agents is to non-specifically dissolve the cell membrane, which damages membrane proteins. They are also toxic or volatile, which is very harmful to the human body and will also reduce the staining intensity of membrane-localized proteins.

[0005] Therefore, it is very necessary to find a new cell permeabilizer that is non-toxic, does not damage cells, and has high staining intensity. Summary of the Invention

[0006] In order to solve at least one of the above problems, the present invention provides a novel cell permeabilizer and its application. The cell permeabilizer can effectively permeabilize adherent and suspended cells and is suitable for different antigen localization. After permeabilization treatment, the cell morphology is normal and there is no nonspecific background signal.

[0007] In order to achieve the above object, the present invention adopts the following technical means: A first aspect of the present invention provides a novel cell permeabilizing agent comprising tea tree oil.

[0008] In some preferred embodiments of the present invention, rutin is further included. By adding rutin, the permeabilization agent is further optimized, cell damage is reduced, and performance is improved.

[0009] Tea tree oil is a pure, natural essential oil extracted from the leaves of Melaleuca alternifolia (Myrtaceae). Its main components, terpinen-4-ol and γ-terpinene, are lipophilic. Rutin, a flavonoid, has strong antioxidant properties. Both are natural substances with minimal cytotoxicity.

[0010] In some embodiments of the present invention, the mass concentration of the tea tree oil is 0.15-0.3%, and the mass concentration of the rutin is 0.05%-0.1%.

[0011] In some embodiments of the present invention, the mass concentration of the tea tree oil is 0.2%, and the mass concentration of the rutin is 0.05%-0.1%.

[0012] A second aspect of the present invention provides the use of tea tree oil / tea tree oil and rutin in the preparation of a cell permeabilizing agent.

[0013] In some embodiments of the present invention, the mass concentration of the tea tree oil is 0.15-0.3%, and further, the mass concentration of rutin compounded with the tea tree oil is 0.05%-0.1%.

[0014] The cell permeabilizing agent is suitable for various cell permeation occasions, including promoting drug penetration.

[0015] The third aspect of the present invention provides a kit comprising the cell permeabilizing agent described in the first aspect.

[0016] In some embodiments, the kit is used for immunofluorescence, immunohistochemistry, in situ hybridization, and other assays. Permeabilization reagents are core tools in these assays, regulating cell membrane permeability to allow antibodies or probes to efficiently access intracellular targets. In immunohistochemistry, the marker can be visualized using a visible light reagent.

[0017] A fourth aspect of the present invention provides the use of tea tree oil / tea tree oil and rutin in preparing a kit.

[0018] In some embodiments of the present invention, the tea tree oil / tea tree oil and rutin are used as cell permeabilizing agents.

[0019] In some embodiments of the present invention, the mass concentration of the tea tree oil is 0.15-0.3% or the tea tree oil is compounded with rutin, and the mass concentration of the rutin is 0.05%-0.1%.

[0020] A fifth aspect of the present invention provides the use of tea tree oil / tea tree oil and rutin in preparing a permeabilized cell sample.

[0021] In some embodiments of the present invention, the cell sample includes wild / transformed tumor cell lines and production cell lines.

[0022] Beneficial effects of the present invention Compared to existing technologies, the present invention has the following advantages: It provides a cell permeabilizer whose active ingredients are the lipophilic tea tree oil and the highly oxidizing rutin. Tea tree oil is a natural plant essential oil that intercalates into the lipid bilayer, altering the cell membrane structure and increasing its permeability to a certain extent. Rutin, a rutinoside of the flavonol compound quercetin, is a flavonoid extracted from buckwheat and ginkgo biloba leaves, which scavenges free radicals and protects cell membrane lipids from oxidative damage. Both are natural extracts and have clear non-toxic properties. The synergistic effect of tea tree oil and rutin allows the permeabilizer to effectively permeate adherent and suspended cells while minimizing cell damage. After permeabilization, the cells exhibit normal morphology and lack nonspecific background signals. Compared to classic permeabilization methods (Triton X-100, saponin, etc.), the permeabilizer in this application achieves comparable or better staining intensity and positive rate, as well as normal cell morphology and lacks nonspecific background signals. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure shows the results of cell immunofluorescence detection under different cell permeabilization agents in Example 1 of the present invention; Figure 2 The figure shows the results of cell immunofluorescence detection under different cell permeabilization agents in Example 2 of the present invention; Figure 3 The figure shows the results of cell immunofluorescence detection under different cell permeabilization agents in Example 3 of the present invention; Figure 4 The figure shows the results of cell immunofluorescence detection under different cell permeabilization agents in Example 4 of the present invention; Figure 5 The figure shows the results of cell immunofluorescence detection under different cell permeabilization agents in Example 5 of the present invention. DETAILED DESCRIPTION

[0024] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0026] The technical solution of the present application will be further described in detail below in conjunction with specific implementation methods.

[0027] Example 1: Effect of tea tree oil on the permeability of plasma- and nuclear-localized single antigens in adherent cells (Hela cells) GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is an enzyme encoded by a housekeeping gene that is widely expressed in eukaryotes. It primarily participates in the glycolysis pathway, catalyzing the oxidation of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate and generating NADH. It is one of the core enzymes in cellular energy metabolism. Its expression level is usually stable, and it is often used as an internal reference protein in molecular experiments. Its subcellular localization is in the cytoplasm and nucleus.

[0028] In this example, cervical cancer cells Hela were selected as test cells to demonstrate the detection effect of different concentrations of tea tree oil as a permeabilizing agent on GAPDH in Hela cells.

[0029] 1. Experimental cells The adherent Hela cells used in the present invention were obtained from Guangzhou Geneo Biotechnology Co., Ltd.

[0030] 2. Cell culture reagents RPMI1640+10%FBS.

[0031] 3. Culture cells and prepare cell slides Hela cells were subjected to routine cell recovery culture and cell passage, and cell slides were prepared after stable cell growth. The coverslips were washed, sterilized at high temperature, and placed in a 6-well plate. 2 mL of Poly-L-lysine solution was added to each well of the 6-well plate to cover the coverslips, and placed in a 37°C incubator for 24 hours to coat the coverslip surface with Poly-L-lysine. Discard the Poly-L-lysine solution and wash twice with 0.01M PBS. B16-F0 cells were seeded on the treated 6-well plate coverslips and cultured in an incubator until the cells covered 60% of the coverslip area. The 6-well plate was removed for cell fixation.

[0032] 4. Cell Fixation Remove the 6-well plate containing the cell slides from the incubator, discard the culture medium, and wash the cells three times with 0.01M PBS. Add 2 mL of 4% paraformaldehyde to each 6-well plate to soak the cell slides and fix the cells at room temperature for 30 minutes.

[0033] 5. Cell permeabilization (1) Experimental groups: Experimental group-1: 0.1wt% tea tree oil; Experimental group-2: 0.2 wt% tea tree oil; Experimental group-3: 0.4 wt% tea tree oil; Control group-1: 0.2wt% Triton X-100; Control group-2: 0.2wt% saponin.

[0034] All the above reagents were prepared using 0.01 M PBS.

[0035] (2) Permeabilization step: After cell fixation, discard the fixative from the 6-well plate and add 0.01M PBS for washing. Wash three times on a decolorizing shaker at low speed for 5 minutes each. Discard the 0.01M PBS and permeabilize the cells by adding 2 mL of the above permeabilization reagent to each well of the 6-well plate. Incubate at room temperature for 15 minutes.

[0036] 6. Sample blocking and antibody binding Discard the permeabilization buffer and wash the cells three times with 0.01M PBS for 5 minutes each. Block the samples by adding 2 mL of 5% skim milk (prepared in 0.01M PBS) to each well of a 6-well plate and incubate at room temperature for 1 hour. After blocking, incubate the cells with the corresponding primary antibody overnight at 4°C. Remove the 6-well plate, discard the primary antibody, and wash the cells six times with 0.01M PBS for 5 minutes each. Incubate the cells with the secondary antibody (fluorescein-labeled) at room temperature for 3 hours in the dark. Discard the secondary antibody and wash the cells six times with 0.01M PBS for 5 minutes each in the dark.

[0037] Rabbit Anti-GAPDH (Abcam, ab9485) was used as the primary antibody, and goat anti-rabbit IgG (CY5 labeled, Abcam, ab6564) was used as the secondary antibody. They were diluted with 2% BSA (0.01M PBS) for later use. The dilution concentrations were based on the instructions for use of the primary and secondary antibodies.

[0038] 7. Nuclear staining, sealing, and testing After the cells are bound to the antibody, incubate them with 1 µg / mL DAPI (in 0.01 M PBS) for 15 minutes at room temperature in a dark place to stain the nuclei. Discard the DAPI staining solution and wash the sample three times with 0.01 M PBS. Place 15 µL of anti-fading mounting medium onto the coverslipped cells. Finally, place the coverslip upside down on a clean glass slide to complete the immunofluorescence sample preparation. Store at 4°C in the dark. Observe the prepared sample using a fluorescence microscope, and capture and output images using software (NIS Elements Viewer).

[0039] 8. The results are as follows Figure 1 shown.

[0040] DAPI is a fluorescent dye that can strongly bind to DNA and can penetrate the cell membrane to stain the cell nucleus. Therefore, it is often used to guide the position of the cell nucleus in immunofluorescence experiments (the cell nucleus is blue when there is no positive overlap). Figure 1 The results show: (1) After permeabilization with 0.2wt% Triton X-100, TTF-1 was specifically recognized by the CY5-labeled antibody and localized in the cytoplasm and nucleus (after merging, red was superimposed on blue, and the nucleus was stained purple; the cytoplasm was not superimposed and was stained red). The staining intensity was strong, the cell structure was clear, there was no shrinkage, and a small amount of cell debris was present. This shows that after permeabilization with 0.2wt% TritonX-100, GAPDH antigen in Hela cells can be well detected and localized, but it has a certain degree of damage to the cells.

[0041] (2) After permeabilization with 0.2 wt% saponin, GAPDH was localized in the cytoplasm and nucleus, the staining intensity decreased, the cell structure was intact, and there was no cell debris. This indicates that the permeabilization effect of 0.2 wt% saponin was poor.

[0042] (3) After permeabilization with 0.2 wt% tea tree oil, the staining results and cell structure were consistent with those of 0.2 wt% Triton X-100, and a small amount of cell debris was also present.

[0043] (4) After permeabilization with 0.1wt% tea tree oil, the staining results were consistent with those with 0.2wt% saponin, and the permeabilization effect was poor.

[0044] (5) After permeabilization with 0.4 wt% tea tree oil, a large amount of cell fragments appeared, indicating that high concentrations of tea tree oil can damage cell structure.

[0045] In summary, 0.2wt% tea tree oil and 0.2wt% Triton X-100 have the same permeability effect on the plasma and nuclear localized single antigens in adherent cells, but both have a certain degree of cell destruction.

[0046] Example 2: The synergistic effect of tea tree oil and rutin on the permeability of plasma- and nuclear-localized single antigens in adherent cells (Hela cells) In this example, cervical cancer cells Hela were selected as test cells to demonstrate the detection effect of 0.2 wt % tea tree oil in combination with different concentrations of rutin as a permeabilizing agent on GAPDH in Hela cells.

[0047] 1. Experimental cells The adherent Hela cells used in the present invention were obtained from Guangzhou Geneo Biotechnology Co., Ltd.

[0048] 2. Cell culture reagents RPMI1640+10%FBS.

[0049] 3. Culture cells and prepare cell slides Hela cells were subjected to routine cell recovery culture and cell passage, and cell slides were prepared after stable cell growth. The coverslips were washed, sterilized at high temperature, and placed in a 6-well plate. 2 mL of Poly-L-lysine solution was added to each well of the 6-well plate to cover the coverslips, and placed in a 37°C incubator for 24 hours to coat the coverslip surface with Poly-L-lysine. Discard the Poly-L-lysine solution and wash twice with 0.01M PBS. B16-F0 cells were seeded on the treated 6-well plate coverslips and cultured in an incubator until the cells covered 60% of the coverslip area. The 6-well plate was removed for cell fixation.

[0050] 4. Cell Fixation Remove the 6-well plate containing the cell slides from the incubator, discard the culture medium, and wash the cells three times with 0.01M PBS. Add 2 mL of 4% paraformaldehyde to each 6-well plate to soak the cell slides and fix the cells at room temperature for 30 minutes.

[0051] 6. Cell Permeabilization (1) Experimental groups: Experimental group-1: 0.2 wt% tea tree oil; Experimental group-2: 0.2wt% tea tree oil, 0.01wt% rutin; Experimental group-3: 0.2wt% tea tree oil, 0.05wt% rutin; Experimental group-4: 0.2wt% tea tree oil, 0.1wt% rutin; Experimental group-5: 0.2wt% tea tree oil, 0.2wt% rutin; Experimental group-6: 0.1 wt% rutin; Control group-1: 0.2wt% Triton X-100; Control group-2: 0.2wt% saponin.

[0052] All the above reagents were prepared using 0.01 M PBS.

[0053] (2) Permeabilization step: After cell fixation, discard the fixative solution from the 6-well plate and add 0.01M PBS for washing. For washing, place the plate on a decolorizing shaker at low speed for 5 minutes each wash, three times. Discard the 0.01M PBS and add 2mL of the above permeabilization reagent to each well of the 6-well plate to permeabilize the cells. Incubate at room temperature for 15 minutes.

[0054] 6. Sample blocking and antibody binding Discard the permeabilization buffer and wash the cells three times with 0.01M PBS for 5 minutes each. Block the samples by adding 2 mL of 5% skim milk (prepared in 0.01M PBS) to each well of a 6-well plate and incubate at room temperature for 1 hour. After blocking, incubate the cells with the corresponding primary antibody overnight at 4°C. Remove the 6-well plate, discard the primary antibody, and wash the cells six times with 0.01M PBS for 5 minutes each. Incubate the cells with the secondary antibody (fluorescein-labeled) at room temperature for 3 hours in the dark. Discard the secondary antibody and wash the cells six times with 0.01M PBS for 5 minutes each in the dark.

[0055] Rabbit Anti-GAPDH (Abcam, ab9485) was used as the primary antibody, and goat anti-rabbit IgG (CY5 labeled, Abcam, ab6564) was used as the secondary antibody. They were diluted with 2% BSA (0.01M PBS) for later use. The dilution concentrations were based on the instructions for use of the primary and secondary antibodies.

[0056] 7. Nuclear staining, sealing, and testing After the cells are bound to the antibody, incubate them with 1 µg / mL DAPI (in 0.01 M PBS) for 15 minutes at room temperature in a dark place to stain the nuclei. Discard the DAPI staining solution and wash the sample three times with 0.01 M PBS. Place 15 µL of anti-fading mounting medium onto the coverslipped cells. Finally, place the coverslip upside down on a clean glass slide to complete the immunofluorescence sample preparation. Store at 4°C in the dark. Observe the prepared sample using a fluorescence microscope, and capture and output images using software (NIS Elements Viewer).

[0057] 8. The results are as follows Figure 2 shown.

[0058] Figure 2 The results show: (1) After permeabilization of experimental group 1, experimental group 2, and control group 1, GAPDH was subcellularly localized in the cytoplasm and nucleus, with strong staining intensity and a small amount of cell debris.

[0059] (2) After permeabilization using experimental groups 3 and 4, GAPDH subcellular localization was in the cytoplasm and nucleus, and the staining intensity was consistent with that of experimental group 1 and control group 1. The cell structure was intact and there was no cell debris. This indicates that the addition of 0.05-0.1wt% rutin to 2wt% tea tree oil has a good protective effect on cells, and the staining intensity remains unchanged.

[0060] (3) After permeabilization using experimental group 5 and control group 2, GAPDH subcellular localization was in the cytoplasm and nucleus, the staining intensity decreased, and the permeabilization effect was poor. This indicates that higher concentrations of rutin will excessively enhance the expression of tight junction proteins, maintain cell barrier function, and lead to poor permeabilization effect.

[0061] (4) After permeabilization using experimental group 6, GAPDH subcellular localization was in the cytoplasm and nucleus, and the staining intensity decreased, indicating poor permeabilization effect. This indicates that the permeabilization effect of rutin alone is poor.

[0062] In summary, for the detection and localization of proteins in the cell nucleus, the new permeabilizer composed of 0.2wt% tea tree oil and 0.05-0.1wt% rutin can be used as a good cell permeabilizer in immunofluorescence experiments.

[0063] Example 3: Permeabilization effect of a novel permeabilizer on a nuclear-localized single antigen in suspension cells (Jurkat cells) C-MYC protein is a basic helix-loop-helix leucine zipper transcription factor encoded by the MYC proto-oncogene. It plays a central role in cell proliferation, differentiation, apoptosis, and metabolic regulation, driving cell cycle progression and growth by activating target genes. The protein is primarily localized to the cell nucleus. C-MYC protein is typically highly expressed in Jurkat cells (a human acute T-lymphoblastic leukemia cell line). Jurkat cells maintain their immortalized phenotype by relying on C-MYC-driven cell cycle progression and anti-apoptotic signals. In this example, Jurkat cells were selected as test cells to demonstrate the effectiveness of a novel permeabilization agent for detecting C-MYC in Jurkat cells.

[0064] 1. Experimental cells The Jurkat suspension cells used in the present invention are from Guangzhou Geneo Biotechnology Co., Ltd.

[0065] 2. Cell culture reagents RPMI1640+10%FBS.

[0066] 3. Culture cells and prepare cell slides Jurkat cells were routinely cultured for cell recovery and passaged. After stable cell growth, natural sedimentation smears were prepared. Suspended cells were collected and centrifuged at low speed (800 rpm) for 5 min. The supernatant was discarded, washed with 0.01 M PBS, and resuspended to an appropriate concentration (1-5 × 10 5 / mL), add equal amount of cell suspension evenly to the slide (vortex thoroughly before adding to prevent agglomeration), bake in a 60℃ oven for 5min, and then take out the slide for cell fixation.

[0067] 4. Cell Fixation Use an immunohistochemistry pen to circle the cell position, add 200uL of 4% paraformaldehyde to the glass slide to soak the cells to fix the cells, and fix them at room temperature for 30 minutes.

[0068] 5. Cell permeabilization (1) Experimental groups: Experimental group-1: 0.2wt% tea tree oil, 0.05wt% rutin; Experimental group-2: 0.2wt% tea tree oil, 0.1wt% rutin; Control group-1: 0.2wt% Triton X-100; Control group-2: 0.2wt% saponin.

[0069] All the above reagents were prepared using 0.01 M PBS.

[0070] (2) Permeabilization step: After cell fixation, discard the cell fixative from the slide and add 0.01M PBS for 30-second washes with shaking for three times. Discard the 0.01M PBS and add 200 μL of the above permeabilization agent to the slide to permeabilize the cells. Incubate at room temperature for 15 minutes.

[0071] 6. Sample blocking and antibody binding Discard the cell permeabilization solution and wash three times with 0.01M PBS for 30 seconds each. Block the slides with 200 μL of 5% skim milk (prepared in 0.01M PBS) and incubate at room temperature for 1 hour. After blocking, incubate the slides with the corresponding primary antibody and cells overnight at 4°C. Remove the slides, discard the primary antibody, and wash six times with 0.01M PBS for 30 seconds each. Incubate the slides with the secondary antibody (fluorescein-labeled) and cells for 3 hours at room temperature in the dark. Discard the secondary antibody and wash six times with 0.01M PBS for 30 seconds each, protecting from light.

[0072] The specific antibody Rabbit Anti-C-MYC (Hangzhou Bailing Biotechnology Co., Ltd., BX50281) was used as the primary antibody, and goat anti-rabbit IgG (CY5 labeled, Abcam, ab6564) was used as the secondary antibody. They were diluted with 2% BSA (prepared in 0.01M PBS) for use. The dilution concentrations were referred to the instructions of the primary and secondary antibodies used.

[0073] 7. Nuclear staining, sealing, and testing After the cells are bound to the antibody, incubate them with 1 µg / mL DAPI (in 0.01 M PBS) for 15 minutes at room temperature in a dark place to stain the nuclei. Discard the DAPI staining solution and wash the sample three times with 0.01 M PBS. Place 15 µL of anti-fading mounting medium onto the coverslipped cells. Finally, place the coverslip upside down on a clean glass slide to complete the immunofluorescence sample preparation. Store at 4°C in the dark. Observe the prepared sample using a fluorescence microscope, and capture and output images using software (NIS Elements Viewer).

[0074] 8. The results are as follows Figure 3 shown.

[0075] Figure 3 The results showed that: (1) After permeabilization using experimental groups 1 and 2, C-MYC subcellular localization was in the cell nucleus, with strong staining intensity, intact cell structure, and no cell debris.

[0076] (2) After permeabilization using control group 1, C-MYC subcellular localization was in the cytoplasm, and the staining intensity was consistent with that of experimental group 1, with a small amount of cell debris present.

[0077] (3) After permeabilization using control group 2, C-MYC subcellular localization was in the cell nucleus, the staining intensity decreased, the cell structure was intact, and there was no cell debris.

[0078] In summary, for the detection and localization of proteins in the cell nucleus, the new permeabilizer composed of 0.2wt% tea tree oil and 0.05-0.1wt% rutin can be used as a good cell permeabilizer in immunofluorescence experiments.

[0079] Example 4: Permeabilization effect of a novel permeabilizer on plasma-localized single antigens in adherent cells (Reh cells) Bcl-2 family proteins are regulators of apoptosis (programmed cell death) and cell survival-promoting factors. They are localized to the mitochondrial membrane, smooth endoplasmic reticulum, and continuous perinuclear membrane and are widely expressed in embryonic tissues. Among lymphocytes, Bcl-2 is highly expressed in T cells, pre-B cells, and mature B cells. Reh cells are a cell line derived from patients with acute lymphoblastic leukemia (ALL). They have a lymphoblastoid morphology and are precursor B cells. Their cytoplasm contains a high abundance of Bcl-2. In this example, Reh cells were selected as test cells to demonstrate the permeabilization effect of a novel permeabilizer on a plasma-localized single antigen in suspended cells (Reh cells).

[0080] 1. Experimental cells The suspension cells Reh used in the present invention are from Guangzhou Keluojie Biotechnology Co., Ltd.

[0081] 2. Cell culture reagents RPMI1640+10%FBS.

[0082] 3. Culture cells and prepare cell slides The Reh cells were subjected to routine cell recovery culture and cell passage, and cell slides were prepared after stable cell growth. The coverslips were washed, sterilized at high temperature, and placed in a 6-well plate. 2 mL of Poly-L-lysine solution was added to each well of the 6-well plate and covered the coverslip. The plates were placed in a 37°C incubator for 24 hours to coat the coverslip surface with Poly-L-lysine. The Poly-L-lysine solution was discarded and the plates were washed twice with 0.01M PBS. B16-F0 cells were inoculated on the treated 6-well plate coverslips and cultured in an incubator until the cells covered 60% of the coverslip area. The 6-well plate was then removed for cell fixation.

[0083] 4. Cell Fixation Remove the 6-well plate containing the cell slides from the incubator, discard the culture medium, and wash the cells three times with 0.01M PBS. Add 2 mL of 4% paraformaldehyde to each 6-well plate to soak the cell slides and fix the cells at room temperature for 30 minutes.

[0084] 5. Cell permeabilization (1) Experimental groups: Experimental group-1: 0.2wt% tea tree oil, 0.05wt% rutin; Experimental group-2: 0.2wt% tea tree oil, 0.1wt% rutin; Control group-1: 0.2wt% Triton X-100; Control group-2: 0.2wt% saponin.

[0085] All the above reagents were prepared using 0.01 M PBS.

[0086] (2) Permeabilization step: After cell fixation, discard the fixative from the slides and add 0.01M PBS for washing. For washing, place the well plate on a decolorizing shaker and shake horizontally at low speed for 5 minutes each time, three times. Discard the 0.01M PBS and add 1 mL of the above permeabilization reagent to each well of the slides to permeabilize the cells. Incubate at room temperature for 15 minutes.

[0087] 6. Sample blocking and antibody binding Discard the permeabilization buffer and wash three times with 0.01M PBS for 5 minutes each. Block the slides with 1 mL of 5% skim milk (prepared in 0.01M PBS) per well and incubate at room temperature for 1 hour. After blocking, incubate the cells with the corresponding primary antibody overnight at 4°C. Remove the 6-well plate, discard the primary antibody, and wash the slides six times with 0.01M PBS for 5 minutes each. Incubate the cells with the secondary antibody (fluorescein-labeled) for 3 hours at room temperature in the dark. Discard the secondary antibody and wash the slides six times with 0.01M PBS for 5 minutes each, protecting the slides from light.

[0088] Rabbit Anti-Bcl-2 (Hangzhou Bailing Biotechnology Co., Ltd., BX50167) was used as the primary antibody, and goat anti-rabbit IgG (FITC-labeled, Abcam, ab6717) was used as the secondary antibody. They were diluted with 2% BSA (0.01 M PBS) for use. The dilution concentrations were referred to the instructions of the primary and secondary antibodies used.

[0089] 7. Nuclear staining, sealing, and testing After the cells are bound to the antibody, incubate them with 1 µg / mL DAPI (in 0.01 M PBS) for 15 minutes at room temperature in a dark place to stain the nuclei. Discard the DAPI staining solution and wash the sample three times with 0.01 M PBS. Place 15 µL of anti-fading mounting medium onto the coverslipped cells. Finally, place the coverslip upside down on a clean glass slide to complete the immunofluorescence sample preparation. Store at 4°C in the dark. Observe the prepared sample using a fluorescence microscope, and capture and output images using software (NIS Elements Viewer).

[0090] 8. The results are as follows Figure 4 shown.

[0091] Figure 4 The results showed that: (1) After permeabilization using experimental groups 1 and 2, Bcl-2 was located in the cytoplasm with strong staining intensity, and the cell structure was intact without cell debris.

[0092] (2) After permeabilization using control group 1, Bcl-2 was localized in the cytoplasm, and the staining intensity was consistent with that of experimental group 1, with a small amount of cell debris present.

[0093] (3) After permeabilization using control group 2, Bcl-2 was localized in the cytoplasm, the staining intensity decreased, the cell structure was intact, and there was no cell debris.

[0094] In summary, for the detection and localization of proteins in the cell cytoplasm, the new permeabilizer composed of 0.2wt% tea tree oil and 0.05wt% rutin can be used as a good cell permeabilizer in immunofluorescence experiments.

[0095] Example 5: Permeabilization effect of a novel permeabilizer on a single membrane-localized antigen in suspended cells (Jurkat cells) The CD19 protein is a B-cell-specific surface antigen and a member of the immunoglobulin superfamily. It promotes B-cell activation, proliferation, and differentiation by enhancing BCR signaling and is involved in the regulation of B-cell development (from pre-B cells to mature B cells) and antibody production. In RAJI cells (a human Burkitt's lymphoma B-cell line), CD19 is highly expressed and subcellularly localized to the cell membrane. In this example, RAJI cells were used as test cells to demonstrate the effectiveness of the novel permeabilization agent for detecting CD19 in RAJI cells.

[0096] 1. Experimental cells The suspension cells RAJI used in the present invention are from Guangzhou Geneo Biotechnology Co., Ltd.

[0097] 2. Cell culture reagents RPMI1640+10%FBS.

[0098] 3. Culture cells and prepare cell slides RAJI cells were routinely cultured for cell recovery and passaged. After stable cell growth, natural sedimentation smears were prepared. Suspended cells were collected and centrifuged at low speed (800 rpm) for 5 min. The supernatant was discarded, washed with 0.01 M PBS, and resuspended to an appropriate concentration (1-5 × 10 5 / mL), add equal amount of cell suspension evenly to the slide (vortex thoroughly before adding to prevent agglomeration), bake in a 60℃ oven for 5min, and then take out the slide for cell fixation.

[0099] 4. Cell Fixation Use an immunohistochemistry pen to circle the cell position, add 200uL of 4% paraformaldehyde to the glass slide to soak the cells to fix the cells, and fix them at room temperature for 30 minutes.

[0100] 5. Cell permeabilization (1) Experimental groups: Experimental group-1: 0.2wt% tea tree oil, 0.05wt% rutin; Experimental group-2: 0.2wt% tea tree oil, 0.1wt% rutin; Control group-1: 0.2wt% Triton X-100; Control group-2: 0.2wt% saponin.

[0101] All the above reagents were prepared using 0.01 M PBS.

[0102] (2) Permeabilization step: After cell fixation, discard the cell fixative from the slide and add 0.01M PBS for 30-second washes with shaking for three times. Discard the 0.01M PBS and add 200 μL of the above permeabilization agent to the slide to permeabilize the cells. Incubate at room temperature for 15 minutes.

[0103] 6. Sample blocking and antibody binding Discard the cell permeabilization solution and wash three times with 0.01M PBS for 30 seconds each. Block the slides with 200 μL of 5% skim milk (prepared in 0.01M PBS) and incubate at room temperature for 1 hour. After blocking, incubate the slides with the corresponding primary antibody and cells overnight at 4°C. Remove the slides, discard the primary antibody, and wash six times with 0.01M PBS for 30 seconds each. Incubate the slides with the secondary antibody (fluorescein-labeled) and cells for 3 hours at room temperature in the dark. Discard the secondary antibody and wash six times with 0.01M PBS for 30 seconds each, protecting from light.

[0104] Rabbit Anti-CD19 (Hangzhou Bailing Biotechnology Co., Ltd., BX50041) was used as the primary antibody, and goat anti-rabbit IgG (FITC-labeled, Abcam, ab6717) was used as the secondary antibody. They were diluted with 2% BSA (0.01 M PBS) for use. The dilution concentrations were referred to the instructions of the primary and secondary antibodies used.

[0105] 7. Nuclear staining, sealing, and testing After the cells are bound to the antibody, incubate them with 1 µg / mL DAPI (in 0.01 M PBS) for 15 minutes at room temperature in a dark place to stain the nuclei. Discard the DAPI staining solution and wash the sample three times with 0.01 M PBS. Place 15 µL of anti-fading mounting medium onto the coverslipped cells. Finally, place the coverslip upside down on a clean glass slide to complete the immunofluorescence sample preparation. Store at 4°C in the dark. Observe the prepared sample using a fluorescence microscope, and capture and output images using software (NIS Elements Viewer).

[0106] 8. The results are as follows Figure 5 shown.

[0107] Figure 5 The results showed that: (1) After permeabilization of experimental groups 1 and 2 and control group 2, CD19 subcellular localization was at the cell membrane, with strong staining intensity, intact cell structure, and no cell debris.

[0108] (2) After permeabilization using control group 1, CD19 subcellular localization was at the cell membrane, and the staining intensity was consistent with that of experimental group 1, with a small amount of cell debris present.

[0109] In summary, for the detection and localization of proteins on the cell membrane, the new permeabilizer composed of 0.2wt% tea tree oil and 0.05-0.1wt% rutin can be used as a good cell permeabilizer in immunofluorescence experiments.

[0110] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.

Claims

1. A novel cell permeabilizing agent, characterized in that: Includes tea tree oil.

2. A novel cell permeabilizing agent according to claim 1, characterized in that: Also includes rutin.

3. A novel cell permeabilizing agent according to claim 1 or 2, characterized in that: The mass concentration of the tea tree oil is 0.15-0.3%.

4. A novel cell permeabilizing agent according to claim 3, characterized in that: The mass concentration of the rutin is 0.05%-0.1%.

5. Application of tea tree oil / tea tree oil and rutin in the preparation of cell permeabilization agents.

6. The use according to claim 5, characterized in that: The mass concentration of the tea tree oil is 0.15-0.3%, and the mass concentration of the rutin is 0.05%-0.1%.

7. A kit, characterized in that: The novel cell permeabilizing agent comprises the novel cell permeabilizing agent according to any one of claims 1 to 4.

8. Use of tea tree oil / tea tree oil and rutin in preparing a kit.

9. Application of tea tree oil / tea tree oil and rutin in the preparation of permeabilized cell samples.

10. The use according to claim 9, characterized in that: The cell samples include wild / transformed tumor cell lines and production cell lines.

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