Freeze/thawing cells

Use conditions that minimize lysis, which can contribute to elevated background. Ensure Digitonin is optimized for cell types.

Freezing cells

  1. Count cells and confirm viability, integrity, and morphology using Trypan Blue staining. Spin cells 600 x g, 3 min, room temperature (RT).
  2. 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.
  3. Slowly freeze aliquots (-1˚C per minute) in an isopropanol-filled chiller in a -80°C freezer (e.g. “Mr. Frosty”).

Thawing cells

  1. 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.
  2. When cells are almost thawed, remove from 37˚C and pipette to fully thaw cells.
  3. Spin cells at 600 x g, 3 min, RT. Pipette to remove supernatant.
  4. 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.
  5. Continue to ConA bead binding (Protocol: Section III).

Did this answer your question? Thanks for the feedback There was a problem submitting your feedback. Please try again later.