Intracellular flow cytometry staining protocol (Transcription Factor Staining Buffer Set 1:50)

Protocol for immunostaining of intracellular markers for flow cytometric analysis using the Transcription Factor Staining Buffer Set.  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.
  • Always prepare Fixation/Permeabilization Solution freshly as described in the data sheet of the Transcription Factor Staining Buffer Set (# 130-122-981).
  1. Wash up to 106 cells by adding 1–2 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
  2. (Optional) Stain cell surface antigens that are sensitive to fixation with appropriate antibodies according to the manufacturer's recommendations. Then wash cells by adding 1–2 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
  3. Resuspend up to 106 cells in 1 mL of cold, freshly prepared Fixation/Permeabilization Solution.
  4. Mix well and incubate for 30 minutes in the dark in the refrigerator (2−8 °C).
  5. Wash cells by adding 1 mL of cold buffer per 106 cells and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
  6. Wash cells by adding 1 mL of cold 1× Permeabilization Buffer per 106 cells and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
  7. Resuspend up to 106 nucleated cells in 98 μL of cold 1× Permeabilization Buffer.
  8. Add 2 μL of the antibody.
  9. Mix well and incubate for 30 minutes in the dark in the refrigerator (2−8 °C).
  10. Wash cells by adding 1 mL of cold 1× Permeabilization Buffer per 106 cells and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
  11. Resuspend cell pellet in a suitable amount of buffer for analysis.
  • Do not use propidium iodide (PI) or 7-AAD staining.
  • Due to fixation and permeabilization, cells are smaller than viable cells. Thus, FSC/SSC settings of the flow cytometer might need to be adjusted.

Materials