Cell Surface Staining for Live Cells using Flow Cytometry

1. Collect cells and wash with PBS.
2. Count cells.
3. Pellet cells so that 50 μL will contain the desired number of cells for staining.
   1. Typically, 5 × 10⁵ – 1 × 10⁶ cells are used per sample.
4. Wash cells in flow staining buffer.
5. Resuspend cells at 1 × 10⁷ cells/mL in flow staining buffer containing blocking agent.
   1. Option 1: Fc block: Block cells using IgG Fc Fragment (P80-104).
      1. Add 1 μg (1 μL) for each 10⁶ cells.
      2. Incubate at room temperature for 10 minutes.
   2. Option 2: Normal Serum: If performing indirect staining, use serum from the animal the secondary antibody is derived. (Often goat serum or calf serum is used.)
      1. Dilute serum to a final concentration of 5% normal serum.
      2. Incubate on ice for 30 minutes.
6. Dilute primary antibody in 50 μL of flow staining buffer.
7. Add 50 μL of antibody solution with the 50 μL of cell suspension for a final volume of 100 μL.
8. Incubate on ice for 30 minutes. If the primary antibody is conjugated to a fluorophore, make sure the incubation is performed in the dark.
9. Wash cells with flow staining buffer.
10. Repeat wash.
11. If using an unconjugated primary antibody:
    1. Dilute your secondary antibody in 100 μL/sample.
    2. Add 100 μL of secondary antibody to each sample.
    3. Incubate on ice for 30 minutes.
    4. Wash cells twice with flow staining buffer.
12. Resuspend cells in at least 200 μL flow staining buffer.
13. Analyze cells on the flow cytometer as soon as possible.

Notes:

1. All centrifugations were performed at 300 × g for 5 minutes.
2. If it will be an hour or more before you can analyze your cells, you may need to fix the cells.