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<p><strong>Intracellular cytokine staining for flow cytometry (mouse)</strong></p> <p><span><strong>Overview</strong></span></p> <p><span>Staining for intracellular cytokines, followed by flow cytometry analysis, can provide single-cell information about a cell population that one cannot obtain from surface staining or ELISA (enzyme-linked immunosorbent assay). </span></p> <p><span><strong>Materials</strong></span></p> <p><span>Supplies <br>96-well V-bottom plate <br>12 x 75mm Falcon round-bottom tubes </span></p> <p><span><strong>Reagents</strong> </span></p> <p><span>DMEM-10 <br>1L DMEM (with 4.5g/L glucose, L-glutamine, sodium pyruvate; from Mediatech, catalog# 10-013-CM) <br>100mL fetal bovine/calf serum <br>10mL 100x PSG (penicillin G sodium, streptomycin sulfate, L-glutamine; from Gibco, catalog# 10378-016) <br>10mL HEPES buffer solution (1M) <br>10mL non-essential amino acids solution (10mM, 100X; Gibco catalog# 11140) <br>1mL 2-mercaptoethanol (1000X) <br>sterilize using 0.2μm filter; store at 4° <br>stimulation solution <br>5mL complete DMEM <br>2.5μL of 200µg/ml PMA (phorbol 12-myristate-13-acetate) <br>1.35μL of 10mM ionomycin <br>20μL of 10mg/ml brefeldin A <br>chemicals will stick to plastic; make just before use, and discard solution afterwards <br>FACS buffer <br>97mL PBS (phosphate buffered solution) <br>3% fetal bovine/calf serum (i.e. 3mL) <br>0.1% sodium azide (i.e. 100μL; optional, especially if you do not plan to store the buffer after use) <br>PBS (phosphate buffer solution) <br>BD Cytofix/Cytoperm solution (or 4X eBioscience permeabilization solution and eBioscience permeabilization diluent) <br>BD Perm/Wash buffer (or 10X eBioscience permeabilization buffer) <br>antibodies <br>Fc block (2.4G2) <br>fluorochrome-linked surface markers (e.g. CD3e, CD4, CD8) <br>fluorochrome-linked cytokine antibodies (e.g. IFN-gamma, IL-12) <br></span> </p><p><span><strong>Equipment</strong> </span></p> <p><span>P200 pipette <br>P200 or P300 multichannel pipette (optional) <br>flow cytometer <br></span> </p><p><span><strong>Procedure</strong> </span></p> <p><span>1. Dilute single-cell suspensions to 10x10^6 cells/mL in complete DMEM. <br>2. Add 100µl cells per well (do not forget to make wells for your staining controls). <br>3. Add 100µl stimulation mix (final concentrations: PMA = 50 ng/ml, ionomycin = 1µg/ml, brefeldin A = 10µg/ml) <br>4. Incubate 37° for 4 hours. <br>5. Spin plate at 800 x g, 3 minutes, at 4°. <br>6. Wash 3 times with cold PBS, spinning as in step 5. <br>7. Resuspend in 100µl Fc block (recommended dilution: 1:1000 in FACS buffer). Incubate on ice, 10 minutes. Spin. <br>8. Resuspend in 100µl surface antibody mixture (recommend dilution: 1:200 in FACS buffer). Incubate on ice, 20 minutes in the dark. Spin. <br>9. Wash once with cold PBS. <br>Note: for steps 10 through 13, either use all of BD reagents or all of eBioscience reagents. Do not mix-and-match. <br>10. Resuspend in 200µl of either BD Cytofix/CytoPerm solution OR 1X eBioscience permeabilization solution. Incubate on ice, 30 minutes in the dark. Spin 1500 x g, 5 minutes, 4°. <br>11. Wash once with 200µl either BD Perm/Wash buffer OR 1X eBioscience permeabilization buffer. Spin as in step 10. <br>12. Resuspend in 100µl cytokine stain (recommended dilution: 1:100 in 1X Perm/Wash OR permeabilisation buffer). Incubate on ice, 30 minutes in the dark. Spin as in step 10. <br>13. Wash twice with BD Perm/Wash OR eBioscience permeabilization buffer, spinning as in step 10. <br>14. Resuspend cells in 100-200µl FACS buffer and transfer to Falcon round-bottom tubes for acquisition on a flow cytometer.</span></p> <p><span><strong>Notes</strong> </span></p> <p><span>If you make the FACS buffer fresh every time, there is no need to add sodium azide to the buffer. <br>All antibody concentrations here are only recommendations. You should titrate the antibody concentrations for your specific cell populations. <br>Non-commercial reagents can also be used for this protocol. See Current Protocols, Unit 6.24 [1]. <br>*As you are permeabilizing the cells through this protocol, the cells are not viable. You cannot sort your cells based on intracellular staining</span></p> <p><span><span><strong>References</strong></span></span></p> <ol><li>Current Protocols in Immunology, Unit 6.24: Detection of Intracellular Cytokines by Flow Cytometry.</li> <li>eBioscience, Protocol for IC Staining link (scroll to bottom of page).</li> <li>BD Biosciences, Protocols: Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis.</li> </ol><p><em>Source: OpenWetWare.org</em></p>
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