How to Freeze Bone Marrow MNC

Reagents Required

• Washing Buffer: PBS (Ca2+ and Mg2+ free) with 2mM EDTA and 10% FBS
• CryostorTM CS
• Fetal Bovine Serum (FBS)
• Ice

Equipment Required

• Refrigerated centrifuge
• Biosafety hood (Class II)
• 2mL Cryogenic vials
• Compound microscope
• 1 to 10 mL pipets
• Isopropanol graded freezing containers
• -20oC freezer
• -80oC freezer

Procedure

• Determine the number of vials needed and the concentration of freeze.

• Concentration of cells <1×106 freeze at 1.2ml

• Concentration of cells >1×106 freeze at 1.8ml

• Centrifuge the cells in a 50ml conical tube at 200xg at 4°C for 10 minutes without brake. Remove the supernatant.

• Use Cryostor™ CS5. Resuspend a small amount of Cryostor CS5 with a P1000 pipettman. Bring the cell suspension to the required amount for freezing.

• Aliquot the appropriate amounts of cell suspension in the cryogenic vials for freeze.

• Place vials in isopropanol graded freezing containers and place in a -80 °C freezer. For long-term storage (>1 month), we recommend moving vials to the vapor phase of liquid-nitrogen storage.

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