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Staining of Intracellular & Nuclear Antigens by Flow Cytometry

This protocol describes a fixation and permeabilization method for staining intracellular and nuclear antigens by flow cytometry.
  1. Collect cells and wash with PBS.
  2. Count cells.
  3. Resuspend cells to be 5 × 106 cells/mL.
  4. Fix cells:
    1. Resuspend cells with paraformaldehyde to a final concentration of 2% by combining equal volumes of cell suspension and 4% PFA.
    2. Incubate cells on ice for 15 minutes.
    3. Pellet cells at 300 × g for 5 minutes.
    4. Discard supernatant in appropriate waste container.
  5. Permeabilize cells:
    1. Resuspend cells in 90% methanol at 1 × 107 cells/mL.
    2. Incubate at -20 °C for at least 30 minutes.
    3. Note: Cells can be stored in 90% methanol for an extended period of time.
  6. Aliquot the appropriate amount of cell suspension for your experiment.
    1. Typically, 5 × 105 – 1 × 106 cells are used per sample.
  7. Pellet cells.
    1. Discard supernatant in appropriate waste container.
  8. Wash cells twice in flow staining buffer.
  9. Resuspend cells 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) per 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.
  10. Dilute primary antibody in 50 μL of flow staining buffer.
  11. Add 50 μL of antibody solution with the 50 μL of cell suspension for a final volume of 100 μL.
  12. Incubate on ice for 30 minutes. (If the primary antibody is conjugated to a fluorophore, make sure the incubation is performed in the dark.)
  13. Wash cells with flow staining buffer.
  14. Repeat wash.
  15. 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.
  16. Resuspend cells in at least 200 μL flow staining buffer.
  17. Analyze cells on the flow cytometer as soon as possible.

Notes:

1. All centrifugations were performed at 300 × g for 5 minutes.

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