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STAINING PROTOCOL

PROTOCOL FOR INTRACELLULAR STAINING WITH FIX & PERM®

  1. For each sample to be analyzed, add appropriate volume of the conjugated surface marker(s) and /or isotype control(s).
  2. Pipette appropriate volume of adjusted cells (equivalent to 1 x 1 million cells) into each tube containing the conjugated surface marker or isotype control.
  3. Vortex each tube gently to mix, and incubate for 15 min. in the dark at room temperature.
  4. Add 100µl of Reagent A (Fixation Medium) and incubate for 15 min. at room temperature.
  5. Wash once in 3 ml PBS + 0.1% NaN3 + 5% FBS.
  6. Centrifuge for 5 min. @ 300-350 X g and aspirate supernatant.
  7. To the cell pellet add: 100µl of Reagent B (Permeabilization Reagent) and the recommended volume of the FITC and/or PE conjugated intracellular antibody or the corresponding isotype controls.
  8. Vortex 1-2 seconds and incubate for 20 min. in the refrigerator.
  9. Wash once in 3 ml PBS + 0.1% NaN3 + 5% FBS.
  10. Centrifuge for 5 min. @ 300-350 g and aspirate the supernatant.
  11. Reconstitute with 0.5 ml PBS + 0.1% NaN3 + 5% FBS.
  12. Analyze on a flow cytometer with at least 2 color capability.

METHANOL MODIFICATION

  1. For each sample to be analyzed, add appropriate volume of adjusted cells (equivalent to 1 X 1 million cells) to a 5 ml tube.
  2. Add 100µl of Reagent A (Fixation Medium) and incubate for 2-3 min. at room temperature.
  3. Add 4 ml of precooled absolute methanol (0-4°C) under shaking (vortex).
  4. Incubate for an additional 10 min. @ 0-4°C.
  5. Centrifuge for 5 min. @ 300-350 X g and wash with PBS + 0.1% NaN3 +5% FBS.
  6. Remove supernatant, and to the cell pellet add: 100µl of Reagent B (Permeabilization Reagent) and appropriate volume of the FITC conjugated intracellular antibody or corresponding isotype control.
  7. Vortex at low speed for 1-2 seconds and incubate for 30 min. at room temperature.
  8. Wash once in 3 ml PMS + 0.1% NaN3 +5% FBS.
  9. Centrifuge for 5 min. @ 300-350 g and aspirate the supernatant.
  10. Remove supernatant and suspend cells in sheath fluid for immediate analysis or re suspend cells in 0.5 ml of 1.0% formaldehyde and store at 2-8°C in the dark.
  11. Analyze fixed cells on a flow cytometer within 24 hours.

The methanol modification is recommended for cell cycle antigens such as Ki-67 and PCNA when using FITC conjugated antibodies. It is not recommended when using R-PE conjugated antibodies.

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CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu EMAIL robinson@flowcyt.cyto.purdue.edu