Red Blood Cell Lysis Protocols

实验概要

BioLegend’s  Red Blood Cell (RBC) Lysis Buffer (Cat. No. 420301) has been designed,  formulated, and tested to ensure optimal lysis of RBCs in single cell  suspensions of hematopoietic tissues, such as spleen, as well as RBCs  derived from peripheral blood. RBC Lysis Buffer contains ammonium  chloride, which has been demonstrated to lyse RBCs with a minimum of  harmful effects on lymphocytes.

主要试剂

Cell Staining Buffer (Details: Biolegend, Cat. No. 420201)

RBC Lysis Buffer (Details: Biolegend, Cat. No. 420301)

实验步骤

Method I: 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 1X working concentration  with deionized water and resuspend the pellet in 3-5 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 10-20 ml of 1X PBS.

6.  Spin the cells (350 x g), discard the supernatant, and resuspend the  pellet in the appropriate buffer (e.g., BioLegend Cell Staining Buffer  Cat. No. 420201).

7. Count cells, adjust density, and proceed with cell staining procedures.

Method II: Lysis of Human Peripheral Blood RBCs:

1.  Dilute the 10X RBC 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 1X RBC Lysis Buffer to each tube containing up to 100 µl of whole blood.

3.  Gently vortex each tube immediately after adding the lysing solution.  Incubate at room temperature, protected from light, for 10-15 minutes.

4.  Centrifuge 350 x g for 5 minutes. Aspirate supernatant without  disturbing pellet, and resuspend the pellet in the appropriate buffer  (e.g., BioLegend Cell Staining Buffer, Cat. No. 420201).