Immunofluorescence Staining of Human Cells by Lysed Whole Blood Method
- Add 100 l of well-mixed anticoagulated whole blood to the bottom of a labeled tube.
- Add the appropriate primary antibody to each tube. If using unlabeled antibody, a titration is suggested. Conjugated antibody should be used as directed, usually 20 l per sample.
- Mix well, then incubate in the dark at room temperature for 20-30 minutes.
- Remove tubes from dark chamber and mix each tube well. Add 2 ml of lysing solution to each tube. Individually vortex each tube.
- Incubate at room temperature in the dark for 10-15 minutes.
- Centrifuge for 5 minutes at 1000 rpm (200 x g).
- Remove supernatant by aspiration, vortex, and add 2 mls washing solution to each tube.
- Centrifuge for 5 minutes at 1000 rpm (200 x g).
- Remove supernatant by aspiration.
- If required, add appropriate second step antibody (at optimal concentration) to each tube and vortex gently (if second step antibody is not required, proceed to step 18).
- Incubate in the dark at room temperature for 20-30 minutes.
- Remove from the dark.
- Mix well, then add 2 ml washing buffer to each tube.
- Centrifuge for 5 minutes at 1000 rpm (200 x g).
- Remove supernatant by aspiration and vortex.
- If third step is not required, proceed to step 18. For third step, add Sav-PE to each tube and vortex gently.
- Repeat steps 11-15.
- If you will analyze the same day, add 500 l wash buffer to each tube, vortex, and analyze within 8 hours. If not, add 2% formaldehyde buffer to each tube, vortex, and store in the refrigerator at 2-8º C for up to 36 hours.
Solutions:
Washing Solution: PBS + 0.1% sodium azide + 1% fetal bovine serum.
Formaldehyde buffer: 2% formaldehyde in PBS.
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