Shreffler:RBC Lysis Buffer for Eos

RBC Lysis Buffer recipe (for Eosinophil protocol)

Materials

• NH4Cl (Sigma A0171-100G)
• KHCO3 (Sigma 237205-100G)
• 0.5 M EDTA (Fisher EC 200-449-4)
• 500 ml millipore filter flask (Millipore SCGPU05RE)

Procedure

1. Measure out 500 ml DI water in beaker (slightly less is OK).
2. Bring solution to 0.8% (4 g) NH4Cl (Ammonium Chloride, above Bert’s bench).
3. Bring solution to 0.1 mM EDTA (100 µl of 0.5 M EDTA stock).
4. Bring pH to 7.2 using 2M KHCO3 (Potassium Bicarbonate, in 50 ml conical above Bert’s bench, but if more see recipe below).
5. Filter in 500 ml flask.

• For 2 M KHCO3, add 5 g KHCO3 to 25 ml DI water in 50 ml conical tube.