Freeze/thawing cells
Use conditions that minimize lysis, which can contribute to elevated background. Ensure Digitonin is optimized for cell types.
Freezing cells
- Count cells and confirm viability, integrity, and morphology using Trypan Blue staining. Spin cells 600 x g, 3 min, room temperature (RT).
- Remove supernatant. Resuspend in cell culture media with 10% DMSO and aliquot as desired. EpiCypher typically aliquots 5 million cells for 8 reactions, which allows for ~20% sample loss during freeze/thaw.
- Slowly freeze aliquots (-1˚C per minute) in an isopropanol-filled chiller in a -80°C freezer (e.g. “Mr. Frosty”).
Thawing cells
- When ready to perform CUT&RUN, remove tubes from -80˚C and quickly place on a 37°C block to thaw. Work quickly to avoid cell lysis.
- When cells are almost thawed, remove from 37˚C and pipette to fully thaw cells.
- Spin cells at 600 x g, 3 min, RT. Pipette to remove supernatant.
- Resuspend cells in 105 μL per reaction RT Wash Buffer. Take a 10 μL aliquot to count using Trypan Blue staining. Note that viability may be decreased; focus instead on cell integrity, lysis levels, and total cell counts. If significant sample loss has occurred, spin cells again and resuspend in a smaller volume of Wash Buffer.
- Continue to ConA bead binding (Protocol: Section III).
