Extracellular Staining Protocol

Date Published: July 18, 2023

Materials: Maintain Reagents and buffers at 4°C.

  1. Fluorochrome-conjugated primary antibody
  2. Fluorochrome-conjugated isotype control antibody
  3. Fixation Buffer, ADI-950-011
  4. PBS (Phosphate Buffered Saline), OmniPur 6505
  5. FBS (Fetal Bovine Serum), Gibco 10082
  6. 10 x 75 mm Tubes, VWR 60818-281

Procedure:

  1. Harvest N x 106 cells and wash twice in 20 ml of PBS followed by centrifugation at 400 x g for 7 minutes (N=number of samples to be tested +1 for each isotype control).
  2. Re-suspend cells in N x 1 ml of Fixation Buffer.
  3. Mix well by inverting and incubate for 30 minutes at 4°C.
  4. Mix well by inverting and centrifuge at 400 x g for 7 minutes.
  5. Decant supernatant and resuspend cells in N x 1 ml of PBS.
  6. Centrifuge at 400 x g for 7 minutes.
  7. Decant supernatant and resuspend pellet in N x 50 µl of PBS.
  8. Gently mix by pipette and aliquot the cell suspension in 50 µl aliquots.
  9. Add the appropriate amount of antibody, to the cell aliquots, to achieve the target working concentration.
  10. Gently mix by pipette and incubate for 30 minutes in the dark at 4°C.
  11. Wash by adding 1 ml of PBS.
  12. Gently mix by pipette and centrifuge at 400 x g for 7 minutes.
  13. Decant supernatant and wash in 1 ml of PBS.
  14. Gently mix by pipette and centrifuge at 400 x g for 7 minutes.
  15. Decant supernatant and gently resuspend cell pellet by pipette with 500 µl of PBS containing 1% FBS. Evaluate cell staining on flow cytometer.

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