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. Visit www.cellsignal.com/flowdyes for a 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: 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 August 2019