10X RBC Bulk Lyse Buffer

The pH of the 1X solution should fall within the range of pH 7.1-7.4 (Note: Adjustment of the pH may be necessary.) Warm the 1X solution to room temperature prior to use.

RBC Lysis Buffer is supplied as a 10X solution containing ammonium chloride, potassium carbonate, and EDTA, and should be diluted in deionized water prior to use.

Store RBC lysis buffer between 2°C and 8°C.

Stable for 12 months from the date of manufacture.

Leinco Technologies' Red Blood Cell (RBC) lysis 10X buffer has been designed, formulated, and tested to ensure optimal lysis of RBCs in single-cell suspensions with minimal effects on leukocytes.

Directions For Use With Directly Labeled Antibodies
Lysis of Mouse Spleen RBCs:
1.) Harvest mouse spleen and prepare a single-cell suspension.
2.) Pellet the cells by centrifugation (350 x g); aspirate the supernatant.
3.) Dilute the 10X Red Blood Cell Lysis Buffer to the 1X working concentration with deionized water and resuspend the pellet in 5 ml of 1X Lysis Buffer.
4.) Incubate on ice for 4-5 minutes with occasional shaking.
5.) Stop the reaction by diluting the Lysis Buffer with 20-30 ml of 1X PBS.
6.) Spin the cells (350 x g) and discard the supernatant.
7.) Resuspend the pellet in the appropriate buffer.
8.) Count cells, adjust the density and proceed with cell staining procedures.

Lysis of Human Peripheral Blood RBSs
1.) Dilute the 10X RBC Easy-Lysis Buffer to 1X working concentration with deionized water. Warm the 1X solution to room temperature prior to use.
2.) Add 2.0 ml of RBC Lyse Buffer to each tube containing 100 µl of whole blood with directly labeled the antibody according to the manufacturer's recommended procedures.
3.) Gently vortex the blood immediately after adding 2 ml of the working concentration 1X RBC Lyse buffer and again vortex the cells vigorously. It is important to vortex each tube immediately after adding RBC Lyse Solution.
4.) Incubate cells at room temperature for ten (10) minutes (Note: Time Can Vary based on a sample). Exposure to RBC Easy-Lyse should not exceed 20 minutes.
5.) Centrifuge leukocytes for 5 minutes at 300 - 500 x g. Decant or Aspirate the supernatant and wash the cells with 2 ml of the working concentration of 1 X washing buffer or an appropriate buffer by gently vortexing the cells and centrifuging for 5 minutes at 300 - 500 x g. If flow cytometric analysis is to be performed within an hour and no fixing is desired, resuspend the cells by gently vortexing in 0.5 ml of wash buffer. Cells are now ready for analysis.
6.) If the analysis is to be performed after one hour or if fixing is desired, add 0.5 ml of the working concentration 1X fixative solution to the pelleted cells from step 4. Vortex the cells gently and they are now ready for flow cytometric analysis. Do not fix cells if they are to be used for tissue culture. Fixed cells should be stored at 4^oC and are now stable up to 48 hours for analysis.

Leinco Technologies makes this buffer under optimal conditions and in accordance with our SOP's and ISO9001.2015 Quality System.

1.) Blaha, Michael & DuBose, David (2002). A HUMAN WHOLE BLOOD MODEL FOR SCREENING POTENTIAL VESICANT ANTAGONISTS. Natick, MA: US Army Research Institute of Environmental Medicine.Link