Protocols: Flow Cytometery

Flow Cytometry Protocol for Intracellular Staining

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1. Solutions and Reagents

1.1. 1X PBS

1.2. Blocking buffer: 0.5% BSA in 1X PBS

1.3. 2% paraformaldehyde (1% solution - optional for storing samples)

1.4. 1X FACS permeabilizing solution (BD Biosciences cat. #340973)

1.5. Fluorescently-conjugated secondary antibody (various forms)

2. Protocol

2.1. Collect 1x106 cells/sample.

2.2. Wash cells once with blocking buffer.

2.3. Fix cells with 2% paraformaldehyde and incubate at room temperature for 10 min.

2.4. Wash cells once with blocking buffer.

2.5. Add 0.5 ml 1X FACS permeabilizing solution and incubate at room temperature for 10 min.

2.6. Wash cells once with blocking buffer.

2.7. Incubate cells in blocking buffer for 30 min at room temperature.

2.8. Add primary antibody at the appropriate dilution and incubate for 30 min at room temperature.

2.9. Wash twice with blocking buffer and incubate with fluorescently-conjugated secondary antibody for 30 min at room temperature.

2.10. Wash cells twice with blocking buffer.

2.11. Re-suspend cells in 1X PBS and analyze on flow cytometry. Samples can be kept in 1% paraformaldehyde at 4 ºC overnight.