IMPORTANT: Please see the product-specific Flow Cytometry protocol on the product webpage to confirm whether it may be used with live cells, and for antibody dilution recommendations.
A. Solutions and Reagents
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.
- 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.
NOTE: Count cells using a hemocytometer or alternative method.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to Immunostaining.
NOTE: To prevent binding of human Fc receptors with rabbit IgG, or mouse Fc receptors with rabbit or mouse IgG, pre-incubate live cells with Fc block prior to immunostaining.
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.
- Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay).
- Pellet cells by centrifugation and remove 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 30 min to 1 hr on ice. Protect from light.
- 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 the recommended dilution).
- Incubate for 30 min on ice. Protect from light.
- Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
- Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.
posted February 2011
revised February 2022