Flow Cytometry, Extracellular Epitope Protocol for Mouse Antibodies

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 10X PBS (#12528) to 900 ml water, mix.
2. 4% Formaldehyde, Methanol-free (#47746), Optional
3. 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.
4. Recommended Anti-Mouse secondary antibodies:
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8890
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4410
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (PE Conjugate) #59997

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 for a full listing of cellular dyes validated for use in flow cytometry.

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.

B. Fixation

NOTE: If live cell staining is desired, proceed to Section C.

1. Pellet cells by centrifugation and remove supernatant.
2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
3. Fix for 15 min at room temperature (20-25°C).
4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 1X PBS.

C. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

1. Aliquot desired number of cells into tubes or wells. (Generally, 5x10⁵ to 1x10⁶ 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 the recommended dilution.
4. Incubate for 30-60 minutes at room temperature (20-25°C).
5. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody, prepared in Antibody Dilution Buffer at the recommended dilution.
7. Incubate for 30 minutes at room temperature.
8. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
9. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.