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Intracellular_Staining_Protocol_082615

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16 views3 pages

Intracellular_Staining_Protocol_082615

Uploaded by

albasouthern
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© © All Rights Reserved
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Intracellular Flow Cytometry Staining Protocol:

For the Detection of Intracellular Cytokines and Other Intracellular Targets

Application Notes
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). For cytokine and chemokine detection, it is critical to
include a protein transport inhibitor such as brefeldin A (BioLegend Cat. No. 420601) or monensin (BioLegend
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 targets. 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.

Protocol
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
(BioLegend 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 (BioLegend Cat. No. 420201). 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).
Alternatively, cells can be kept in Cyto-Last™ Buffer (BioLegend Cat. No. 422501) for the storage of cytokine-
producing cells for up to two weeks. 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
4. Dilute 10X Intracellular Staining Perm Wash Buffer (Cat. No. 421002) to 1X in DI water.
5. Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5-10 minutes.
6. Repeat step 5 twice.

BioLegend | San Diego, CA 92121


US Toll-Free Phone: 1-877-Bio-Legend (1-877-246-5343) | Phone: 1-858-455-9588 | Fax: 858.455.9587
Intracellular Staining
7. Resuspend fixed/permeabilized cells in residual Intracellular Staining Perm Wash Buffer and add a
predetermined optimum concentration of fluorophore-conjugated antibody of interest (e.g. PE anti-IFN-γ) or
an appropriate negative control for 20 minutes in the dark at room temperature.
8. Wash 2x with 2 ml of Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5 minutes.
9. If primary intracellular antibody is biotinylated, it will be necessary to perform fluorophore conjugated
Streptavidin incubations and subsequent washes in Intracellular Staining Perm Wash Buffer.
10. 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 fluorophore-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 cytokines or chemokines (e.g. IFN-γ or IL-4), addition of an efficient protein transport inhibitor such
as brefeldin A (BioLegend Cat. No. 420601) or monensin (BioLegend 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 as described
above.
6. Fix, permeabilized, 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 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 coexpress certain levels of cell surface antigen and intracellular cytokine
proteins.

BioLegend | San Diego, CA 92121


US Toll-Free Phone: 1-877-Bio-Legend (1-877-246-5343) | Phone: 1-858-455-9588 | Fax: 858.455.9587
Related Information
1. Assenmacher, M., et al. 1994. Eur. J. Immunol. 24:1097.
2. Elson, L.H., et al. 1995. J. Immunol. 1995. 154:4294.
3. Jung T, et al. 1993. J. Immunol. Methods 159:197.
4. Prussin C., et al. 1995. J. Immunol. Methods 188:117.
5. Vikingsson A., et al. 1994. J. Immunol. Methods 173:219.

Reagent List
1. Cell Staining Buffer (BioLegend Cat. No. 420201)
2. Monensin (BioLegend Cat. No. 420701)
3. RBC Lysis Buffer (BioLegend Cat. No. 420301)
4. Brefeldin A (BioLegend Cat. No. 420601)
5. Fixation Buffer (BioLegend Cat. No. 420801)
6. Intracellular Staining Perm Wash Buffer (BioLegend Cat. No. 421002)
7. Cyto-Last™ Buffer (BioLegend Cat. No. 422501)

BioLegend | San Diego, CA 92121


US Toll-Free Phone: 1-877-Bio-Legend (1-877-246-5343) | Phone: 1-858-455-9588 | Fax: 858.455.9587

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