IMPORTANT: Please see the product-specific Flow Cytometry protocol on the product webpage for appropriate fixation and permeabilization conditions, and recommended antibody dilution.
A. Solutions and Reagents
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Triton™ X-100) #51995, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water, mix.
- 4% Formaldehyde, Methanol-Free (#47746)
- Cell Permeabilization Buffer: Purchase ready-to-use (#39487) or to prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml Antibody Dilution Buffer. Store at 4°C.
- Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
- Recommended secondary antibodies: For detection of unconjugated antibodies, select a secondary antibody that matches the host species of your primary antibody (e.g., rabbit). Click here for an up-to-date list of secondary antibodies approved for flow cytometry.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. View our listing of cellular dyes validated for use in flow cytometry.
B. Fixation and Permeabilization
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or Triton™ X-100. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. Conduct a small-scale experiment if you are unsure.
- Pellet cells by centrifugation and remove supernatant.
- Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
- Fix for 15 min at room temperature (20-25°C).
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container.
- Resuspend cells in approximately 100 µl Cell Permeabilization Buffer per million cells.
- Incubate for 10 minutes at room temperature.
- Proceed with staining or store cells at 4°C in PBS overnight.
NOTE: Count cells using a hemocytometer or alternative method.
- Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay).
- Centrifuge cells and discard supernatant.
- Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
- Incubate for 1 hr at room temperature (20-25°C).
- Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat. If using directly conjugated antibodies, skip to step 9.
- Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at recommended dilution).
- Incubate for 30 minutes at room temperature (20-25°C). Protect from light.
- Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer.