Mouse Erythrocyte Lysing Kit
R&D Systems, part of Bio-Techne | Catalog # WL2000
Key Product Details
Summary for Mouse Erythrocyte Lysing Kit
Kit Summary
Why Remove Erythrocytes?
The lysis of erythrocytes from whole blood is an important initial step in the isolation and analysis of enriched leukocyte preparations. Recovered immune cells can be accurately characterized following red blood cell removal. Lysis of erythrocytes under conditions that do not disrupt lymphocytes or myeloid cells is critical for downstream applications utilizing leukocytes harvested from whole blood.
Reagents Provided
The Mouse Erythrocyte Lysing Kit (Catalog # WL2000) contains the following reagents for the lysis of erythrocytes in splenocyte preparations:
- M-Lyse Buffer Concentrate (10X)
- Wash Buffer Concentrate (10X)
- Fixative Concentrate (10X)
*This kit contains sufficient reagents to process 12.5 x 109 splenocytes.
Stability and Storage
Store all reagents at 20 °C to 25 °C.
Assay Procedure
Refer to the product datasheet for complete product details.
Reagents Provided
The Mouse Erythrocyte Lysing Kit (Catalog # WL2000) contains the following reagents for the lysis of erythrocytes in splenocyte preparations:
- M-Lyse Buffer Concentrate (10X)
- Wash Buffer Concentrate (10X)
- Fixative Concentrate (10X)
This kit contains sufficient reagents to process 12.5 x 109 splenocytes.
Other Supplies Required
- Ficoll-Hypaque™
- Sterile distilled or deionized water
- Sterile centrifuge tubes
- Benchtop centrifuge
- Pipettes and sterile pipette tips
Procedure Overview
R&D Systems Protocol for the Lysis of Erythrocytes Using the Mouse Erythrocyte Lysing Kit
If the cell preparation will be further processed with a T cell enrichment column, complete these steps prior to treating cells with H-Lyse Buffer.
- Stain a single cell suspension of mononuclear cells from a mouse spleen with antibody or antibodies (if performing flow cytometry).
- Wash the cells once with Hanks’ BSS + 10% serum.
- Disrupt the cell pellet by “racking” the tube.
- Add 2 mL 1X M-Lyse Buffer per spleen processed.
- Incubate the cells at room temperature until lysis is complete (10 minutes).
- Wash the leukocytes with 2 mL 1X Wash Buffer.
- Resuspend the cells in 1 mL 1X Wash Buffer.
- Fix the cells with 100 uL 10X Fixative Concentrate if flow cytometry analysis will be delayed more than one hour.
- Or
- Utilize leukocytes for alternate downstream applications
- Prepare a single cell suspension of mononuclear cells.
- Wash the cells with excess sterile PBS.
Technical Hints
- If flow cytometric analysis of the cells will be delayed for more than 1 hour, the cells can be fixed at this time to stabilize the cells for later analysis. This step should be eliminated if cells are to be used for culture.
- Cells should be stored at 2 °C to 8 °C until analysis. Although stained cells will be stable for up to 48 hours, we recommend that flow cytometric analysis be performed as soon as possible.