Whole Blood Lyse/Wash Procedure:

1. Prepare 1X RBC Lysis/Fixation solution by diluting 1 part of BioLegend RBC Lysis/Fixation solution with 9 parts of room temperature dI H₂0.
2. Mix 100 µl of whole blood with fluorochrome-conjugated antibody.
3. Incubate at room temperature, in the dark, for 20 minutes.
4. Add 2.0 mL 1X RBC Lysis/Fixation solution, gently mixing each tube immediately.
5. Incubate at room temperature, in the dark, for 15 minutes.
6. Centrifuge at 350Xg for five minutes.  Discard supernatant.
7. Add 2 mL BioLegend Cell Staining Buffer (Cat No 420201).
8. Centrifuge at 350Xg for five minutes. Discard supernatant.
9. Resuspend cell pellet in 0.3-0.5 mL Cell Staining Buffer.
10. Analyze cells immediately by flow cytometry or store at 4°C, in the dark, for no longer than 12 hours.

Whole Blood Lyse/No Wash Procedure:

1. Prepare 1X RBC Lysis/Fixation solution by diluting 1 part of BioLegend RBC Lysis/Fixation solution with 9 parts room temperature dI H₂0.
2. Mix 50 µl of whole blood with fluorochrome-conjugated antibody.
3. Incubate at room temperature, in the dark, for 20 minutes.
4. Add 0.45 mL 1X RBC Lysis/Fixation solution, gently mixing each tube immediately.
5. Incubate at room temperature, in the dark, for 15 minutes.
6. Analyze samples by flow cytometry.

The 1X Lysis/Fixation solution can be used within 1 month from the date of preparation.

1. Horvath P, et al. 2012. J Infect Dis. PubMed.