胞内细胞因子染色步骤（自BioLegend）

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标题：胞内细胞因子染色步骤（自BioLegend）

Application Notes:

1. Activated cell populations can be prepared from in vivo-stimulated tissues or from in vitro-stimulated cultures (e.g., antigen-specifc activation or mitogen-induced). It is critical to include a protein transport inhibitor such as （布雷菲德菌素A）brefeldin A (Cat. No. 420601) or （莫能菌素）monensin (Cat. No. 420701) in the last 4-6 hours of cell culture activation. The cells can be suspended and distributed to 12 x 75 mm plastic tubes or microwell plates for immunofuorescent staining.
2. Diferent cytokines/chemokines have diferent production peaks. In order to obtain optimal staining signals, the stimulation conditions for each stimulant need to be optimized.
3. Some antibodies recognizing native cell surface markers may not bind to fxed/denatured antigens. For this reason, it is recommended that staining of cell surface antigens be done with live, unfxed cells PRIOR to fxation/permeabilization and staining of intracellular cytokines. Altering the procedure such that cells are fxed prior to staining of cell surface antigens requires that paraformaldehyde-denatured antigen-reactive antibody clones be empirically identifed.

Fixation:

1. If staining intracellular antigens (e.g. IFN-γ or IL-4), frst perform cell surface antigen staining as described in BioLegend’s Cell Surface Immunofuorescence Staining Protocol, then fx cells in 0.5 ml/tube Fixation Bufer (Cat. No. 420801) in the dark for 20 minutes at room temperature.
2. Centrifuge at 350 x g for 5 minutes, discard supernatant.
3. To put the experiment “on hold” at this point for future staining and analysis, wash cells 1x with Cell Staining Bufer. Resuspend cells in Cell Staining Bufer and store cells at 4°C (short term) or in 90\% FCS/10\% DMSO for storage at -80°C (long term, for fxed cells without surface antigen staining). The frequencies of cytokine-producing cells present in activated human PBMC cultures can vary widely due to donor variability. Therefore, cryopreserved
   cells from a single donor are useful for longitudinal studies.

Permeabilization:

1. Dilute 10X Permeabilization Wash Bufer (Cat. No. 421001) to 1X in DI water（去离子水）.
2. Resuspend fxed cells in Permeabilization Wash Bufer and centrifuge at 350x g for 5-10 minutes.
3. Repeat step 5 twice.

Intracellular Staining:

1. Resuspend fxed/permeabilized cells in residual Permeabilization Wash Bufer and add a predetermined optimum concentration of fuorochrome conjugated antibody of interest (e.g. anti-IFN-γ-PE) or an appropriate negative control for 20 minutes in the dark at room temperature.
2. Wash 2x with 2 ml of Permeabilization Wash Bufer and centrifuge at 350 x g for 5 minutes.
3. If primary intracellular antibody is biotinylated, it will be necessary to perform fuorochrome conjugated Streptavidin incubations and subsequent washes in Permeabilization Wash Bufer.
4. Resuspend fxed and intracellularly labeled cells in 0.5 ml Cell Staining Bufer and analyze with appropriate controls.

Note: To confrm specifc anti-cytokine staining, a blocking experiment is recommended in which cells are fxed/permeabilized then preincubated with an excess amount of unlabeled anti-cytokine antibody and/or the recombinant cytokine of interest is preincubated with fuorochrome-conjugated anticytokine antibody before its addition to the cells.

Activation and Intracellular Staining of Whole Blood:

1. Dilute heparinized whole blood 1:1 with sterile appropriate tissue culture medium.
2. At this stage, in vitro cellular stimulation by either antigen or mitogen can be performed. If intending to stain intracellular antigens (e.g. IFN-γ or IL-4), addition of an efcient protein transport inhibitor such as brefeldin A (Cat. No. 420601) or monensin (Cat. No. 420701) is critical. After addition of a suitable cellular activator, aliquot 200 µl of the whole blood cell suspension into 12 x 75 mm plastic tubes and incubate for 4-6 hours in 5\% CO2 at 37°C.
3. Add 2 ml of 1X Red Blood Cell Lysis Bufer (Cat. No. 420301) and incubate for 5-10 minutes at room temperature.
4. Centrifuge at 350 x g for 5 minutes and discard the supernatant.
5. Wash cells 1X with Cell Staining Bufer and perform cell surface immunofuorescent staining.
6. Fix, permeabilize and stain intracellular antigens as described above.

Flow Cytometric Analysis:
Set PMT voltage and compensation using cell surface staining controls. Set quadrant markers based on blocking controls, isotype controls, or unstained cells. For proper fow cytometric analysis, cells stained by this method should be inspected by light microscopy and/or fow light scatter pattern to confrm that they are well dispersed. Bivariate dot plots or probability contour plots can be generated upon data analysis to display the frequencies of and patterns by which individual cells co-express certain levels of cell surface antigen and intracellular cytokine proteins.

Related Information:
1. Jung T, et al. 1993. J. Immunol. Methods 159:197.
2. Vikingsson A., et al. 1994. J. Immunol. Methods 173:219.
3. Prussin C., et al. 1995. J. Immunol. Methods 188:117.
4. Elson, L.H., et al. 1995. J. Immunol. 1995. 154:4294.
5. Assenmacher, M., et al. 1994. Eur. J. Immunol. 24:1097.

Reagent List:
1. Cell Staining Bufer (Cat. No. 420201)
2. Monensin (Cat. No. 420701)
3. RBC Lysis Bufer (Cat. No. 420301)
4. Brefeldin A (Cat. No. 420601)
5. Fixation Bufer (Cat. No. 420801)
6. Permeabilization Wash Bufer (Cat. No. 421001)

seall

倪老师，我现在做小鼠脾细胞的胞内染色，染IL-17、TNFa等几个细胞因子，试了几次效果都不好，有没有最新的好用的固定和破膜试剂推荐？