Intracellular flow cytometry staining protocol (Cell Signaling Buffer Set A 1:50)

Protocol for immunostaining of intracellular markers for flow cytometric analysis using the Cell Signaling Buffer Set A.  Applicable to antibodies with recommended working dilution of 1:50.

Protocol

  • The recommended antibody dilution for labeling of cells and subsequent analysis by flow cytometry is 1:50 for up to 106 cells in final volume of 100 µL.
  • Volumes given below are for up to 106 nucleated cells. When working with fewer than 106 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly.
  • To achieve the appropriate performance for permeabilization of cells, the Permeabilization Buffer A must chill to –20 °C prior to use.
  1. (Optional) Treat cells with appropriate cell activators or stimuli.
    Note: Always include a negative control in the experiment. The sample should be treated in exactly the same manner as the stimulated sample, except for the addition of the stimulus. A positive control should also be included in the experiment.
  2. Fix cells by adding 250 µL Inside Fix to 1 mL cell suspension with 106 cells.
  3. Mix well and incubate for 10 minutes at room temperature.
  4. Centrifuge at 500×g for 5 minutes at 4 °C. Aspirate supernatant carefully.
  5. Resuspend cells and permeabilize the cells by slowly adding 1 mL of ice-cold (–20 °C) Permeabilization Buffer A per 106 cells.
  6. Vortex and place the tubes on ice for 30 minutes.
  7. Wash cells by adding 3 mL of buffer. Centrifuge samples at 500×g for 5 minutes at 4 °C. Aspirate supernatant carefully.
  8. Wash cells by adding 4 mL of buffer and centrifuge at 500×g for 5 minutes at 4 °C. Aspirate supernatant carefully.
  9. Resuspend up to 106 nucleated cells per 98 µL buffer. Add 2 µL of the antibody.
  10. Mix well and incubate for 30 minutes in the dark at room temperature.
  11. Wash cells by adding 1 mL buffer and centrifuge at 500×g for 5 minutes. Aspirate supernatant carefully.
  12. Resuspend cell pellet in a suitable amount of buffer for analysis. Store cells at 2–8 °C in the dark until analysis. Mix well before flow cytometric acquisition.
  • Do not use propidium iodide (PI) or 7-AAD staining.

Materials