Flow Cytometry Live Cell Protocol

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS, add 100 mL of 10X PBS #12528 to 900 mL water. Mix well.
2. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer #9998 in 100 mL 1X PBS. Store at 4°C.
3. 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. Immunostaining

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 off-target binding to Fc receptors, pre-incubate live cells with an Fc blocking buffer prior to immunostaining.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300 g for 1-5 min will be sufficient to pellet the cells.

1. Aliquot desired number of cells into tubes or wells. (Generally, 5 x 10⁵ to 1 x 10⁶ cells per assay).
2. Pellet cells by centrifugation and remove supernatant.
3. Resuspend cells in 100 µL of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
4. Incubate for 30 min to 1 hr on ice. Protect from light.
5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat. If using directly conjugated antibodies, skip to step 9.
6. Resuspend cells in 100 µL of diluted fluorochrome-conjugated secondary antibody, prepared in Antibody Dilution Buffer at a recommended dilution.
7. Incubate for 30 min on ice. Protect from light.
8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
9. Resuspend cells in 200-500 µL of Antibody Dilution Buffer, then analyze on a flow cytometer.

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_{For Research Use Only. Not for Use in Diagnostic Procedures.}