Protocol for RBC Lysis of Mouse Spleen

Monday, May 10, 2004

Description
Prior to using lymphoid tissue cell suspensions for flow cytometric analysis and/or for in vitro functional assays, it is recommended to remove red blood cells. The eBioscience 1X RBC Lysis Buffer (Cat. No. 00-4333) is formulated for optimal lysis of erythrocytes in single cell suspensions of mouse tissues such as spleen and human peripheral blood. The buffer contains ammonium chloride, which lyses red cells with minimal effect on lymphocytes. This step is not necessary when working with mouse thymus and lymph nodes.

When using human peripheral blood for flow cytometric analysis, a red cell lysing step is incorporated into the staining protocol. Click here to go to the protocol for surface staining of human leukocytes.

For lysing human blood in bulk, a 10-20X volume of 1X RBC lysis buffer to total blood is recommended; however, an alternative method for removal of red blood cells when working with human blood is to use isolation of PBMC by Ficoll-Paque.

Procedure
Harvest mouse spleen and prepare a single cell suspension.
Pellet the cells by centrifugation (300-400xg) at 4C and aspirate the supernatant.
Resuspend the pellet in 5 ml/spleen of Lysis Buffer.
Incubate at room temperature for 4-5 minutes with occasional shaking (we have performed this step on ice successfully too).
Stop the reaction by diluting the Lysis Buffer with 20-30 ml of 1X PBS.
Spin the cells (300-400xg) at 4C and resuspend the pellet in the appropriate buffer for use in the next step of your experimental procedure.
Perform a cell count at this time.

Recipes
Materials
1X PBS for recipe
eBioscience 1X RBC Lysis Buffer, Cat. No. 00-4333
50 ml conical tubes
Instruments
Pipettes and pipettors
Centrifuge
Hemacytometer and microscope
Experiment Duration
20 minutes

Supplies

Tips

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Protocol for RBC Lysis of Mouse Spleen