Intracellular Cytokine Staining Protocol

实验概要

A  modification of the basic immunofluorescence staining and flow  cytometric analysis protocol can be used for the simultaneous analysis  of surface molecules and intracellular cytokines at single-cell level.  In this protocol, cells are first activated in vitro, stained for  surface antigens, as in the regular staining protocol, then fixed and  permeabilized to allow for anti-cytokine antibodies to stain  intracellularly. In vitro stimulation of cells is usually required for  detection of cytokines by flow cytometry since cytokine levels are  typically too low in freshly-prepared cells. Stimulation of cells with  the appropriate reagent will depend on the cell type and the  experimental conditions. For example, to stimulate T cells to produce  IFN-gamma, TNF-alpha, IL-2, and IL-4, a combination of PMA (a PKC  activator) and Ionomycin (a calcium ionophore) or anti-CD3 antibodies  are used. To induce IL-6, IL-10 or TNF-alpha production by monocytes,  stimulation of PBMC's with LPS is used.

主要试剂

Cell Staining Buffer [Details: BioLegend Cat. #420201]

Monensin [Details: BioLegend Cat. # 420701]

RBCLysisBuffer[Details: BioLegend Cat. # 420301]

BrefeldinA [Details: BioLegend Cat. # 420601]

FixationBuffer [Details: BioLegend Cat. #420801 ]

PermeabilizationWashBuffer [Details: BioLegend Cat. #421002 ]

实验步骤

1.        Fixation:

1)        If staining intracellular antigens (e.g. IFN-γ or IL-4), first perform  cell surface antigen staining as described in BioLegend’s Cell Surface  Immunofluorescence Staining Protocol, then fix cells in 0.5 ml/tube  Fixation Buffer (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 Buffer. Resuspend cells in  Cell Staining Buffer and store cells at 4°C (short term) or in 90%  FCS/10% DMSO for storage at -80°C (long term, for fixed 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.

2.        Permeabilization:

1)        Dilute 10X Permeabilization Wash Buffer (Cat. No. 421002) to 1X in DI water.

2)        Resuspend fixed cells in Permeabilization Wash Buffer and centrifuge at 350 x g for 5-10 minutes.

3)        Repeat step 5 twice.

3.        Intracellular Staining:

1)         Resuspend fixed/permeabilized cells in residual Permeabilization Wash  Buffer and add a predetermined optimum concentration of fluorochrome  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 Buffer and centrifuge at 350 x g for 5 minutes.

3)         If primary intracellular antibody is biotinylated, it will be necessary  to per- form fluorochrome conjugated Streptavidin incubations and  subsequent washes in Permeabilization Wash Buffer.

4)    Resuspend fixed and intracellularly labeled cells in 0.5 ml Cell Staining Buffer and analyze with appropriate controls.

Note: To  confirm specific anti-cytokine staining, a blocking experiment is  recommended in which cells are fixed/permeabilized then preincubated  with an excess amount of unlabeled anti-cytokine antibody and/or the  recombinant cytokine of interest is preincubated with  fluorochrome-conjugated anti- cytokine 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 efficient 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 Buffer (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 Buffer and perform cell surface immunofluorescent 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  quad- rant markers based on blocking controls, isotype controls, or  unstained cells. For proper flow cytometric analysis, cells stained by  this method should be inspected by light microscopy and/or flow light  scatter pattern to confirm 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.

注意事项

1.         Activated cell populations can be prepared from in vivo-stimulated  tissues or from in vitro-stimulated cultures (e.g., antigen-specific  activation or mitogen- induced). It is critical to include a protein  transport inhibitor such as brefeldin A (Cat. No. 420601) or 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 immunofluorescent staining.

2.         Different cytokines/chemokines have different 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  fixed/denatured antigens. For this reason, it is recommended that  staining of cell surface antigens be done with live, unfixed cells PRIOR  to fixation/permeabilization and staining of intracellular cytokines.  Altering the procedure such that cells are fixed prior to staining of  cell surface antigens requires that paraformaldehyde-denatured  antigen-reactive antibody clones be empirically identified.