Intracellular flow cytometry staining protocol (Inside Stain Kit 1:10, protocol B)

Protocol for immunostaining of intracellular markers for flow cytometric analysis using the Inside Stain Kit. Applicable to antibodies with recommended working dilution of 1:10.

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Protocol

  • The recommended antibody dilution for labeling of cells and subsequent analysis by flow cytometry is 1:10 for up to 106 cells in final volume of 50 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 (e.g. for 2×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
  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 250 µL of buffer.
  4. Add 250 µL of Inside Fix (Inside Stain Kit). Mix well and incubate for 20 minutes in the dark at room temperature.
  5. Centrifuge at 300×g for 5 minutes. Aspirate supernatant carefully.
  6. Wash cells by adding 1 mL of buffer and centrifuge at 300×g for 5 minutes. Aspirate supernatant carefully. 
    ▲Note: Fixed cells may be stored in azide-containing buffer at 2–8 °C for up to 1 week.
  7. (Optional) Stain cell surface antigens that are sensitive to permeabilization 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.
  8. Wash cells by adding 1 mL of Inside Perm (Inside Stain Kit) and centrifuge at 300×g for 5 minutes. Aspirate supernatant carefully.
  9. Resuspend cells in 45 µL of Inside Perm. Add 5 µL of the antibody. 
    ▲Note: For staining with several antibodies in this step, reduce the volume of Inside Perm accordingly. For efficient permeabilization, the volume of Inside Perm should be at least 30% of the overall staining volume.
  10. Mix well and incubate for 10 minutes in the dark at room temperature.
  11. Wash cells by adding 1 mL of Inside Perm and centrifuge at 300×g for 5 minutes. Aspirate supernatant carefully. If biotinylated primary antibody was used, repeat washing step.
  12. (Optional) If biotinylated primary antibody was used, resuspend the cell pellet in Inside Perm and stain with fluorochrome-conjugated Biotin Antibody according to the manufacturer's recommendations.
  13. 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

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