This article gives a brief description of the procedure to lyse, and thus, eliminate red blood cells from flow cytometry samples. Flow cytometry is a common technique used to detect different kinds of antigens and proteins in blood samples, especially for immunophenotyping. The results are much more likely to be accurate and can be analyzed with greater ease if red blood cells within the sample have first been lysed.
The solutions used in this procedure include:
Triton X-100 solution: Freshly prepared 0.12% Triton X-100 in phosphate buffered saline (PBS)
Wash buffer: Either 4% fetal bovine serum or 2% bovine serum albumin in PBS.
Methanol: A stock solution of 50% methanol should be prepared in PBS and it can be stored at a temperature of -20 ºC.
The following steps outline the lysis protocol:
- 65 µL of 10% formaldehyde is added to 100 µl blood to achieve a final concentration of 4% formaldehyde.
- This is incubated at room temperature for ten minutes.
- 1 mL Triton X-100 solution is added for a final concentration of 0.1%.
- This is again incubated at room temperature for 30 minutes.
- 1 mL cold wash buffer is added.
- The sample is placed in the centrifuge and spun, before being resuspended in 1 mL 50% MeOH.
- The suspension is now incubated at 4 ºC for 10 minutes or more.
Red blood cell lysis kits are available commercially, but care should be taken to comply with the manufacturers’ instructions.
Several researchers have written about the effects of detergents on the detection of antigens on red blood cells, both surface and intracellular. The next report deals with the way various detergents, as well as different concentrations of some detergents, affect the lysis of red cells and the resolution of whole blood, both of which were evaluated using light scattering analysis.
It was observed that only three detergents were able to produce significant lysis among red cells after being fixed with 2% formaldehyde for ten minutes at room temperature, namely, Triton X 100, NP-40 and Brij-58. They negatively impacted the light scattering from whole blood cells once the concentration exceeded 0.2%. Below a concentration of 0.1%, the red cells were not lysed fully. The optimal conditions were determined to be incubation of the sample with 0.1% detergent for 30 minutes.
Adapted from: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW and Shankey TV. (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry 67, 4–17.
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