Optimizing cell permeabilization with Digitonin

CUT&RUN uses Digitonin to permeabilize cells and represents a crucial step in the protocol.

Insufficient Digitonin prevents antibody and pAG-MNase from entering the cell, while excess amounts may result in cell lysis. EpiCypher recommends using the minimal amount of Digitonin required to permeabilize >95% of cells. Optimize Digitonin concentrations for each cell type used in CUT&RUN as outlined below.

NOTE: If using nuclei, Digitonin optimization is NOT required. Use 0.01% Digitonin in CUT&RUN buffers to prevent the beads from forming a film on the side of tubes.

Before starting, label your tubes:

  • Label five fresh 1.5 mL tubes with percent Digitonin (see Table, below) and a sixth tube as Control.
  • Label 6 additional tubes with percent Digitonin or as Control. This second set of tubes will be used for cells.

Prepare buffers

  1. Prepare a series of five Cell Permeabilization Buffers using 5% Digitonin and CUT&RUN Wash Buffer (see Protocol: Section I), FRESH on the day of use. Add the appropriate volume of Wash Buffer to each tube as outlined in the Table. Add 10 µL 5% Digitonin to the first tube, labeled 0.05%. Vortex to mix.
  2. Prepare the other four Cell Permeabilization Buffers by serial dilution (see Table). Vortex each buffer to mix and place on ice.
  3. For the Control buffer, prepare 0.05% DMSO in Wash Buffer.
Final % Digitonin 0.05 0.01 0.001 0.0001 0.00001
Volume from previous tube (µL) - 200 100 100 100
Wash buffer (µL) 990 800 900 900 900
5% Digitonin (µL) 10 - - - -

Use above Table to prepare serial dilutions of Digitonin.

Permeabilize cells

  1. Harvest cultured cells for permeabilization testing. To determine the number of cells needed for Digitonin optimization, multiply the number of cells used per CUT&RUN reaction (500,000) x 6.2 (six tubes + 20% excess volume for pipetting errors).
  2. Spin 600 x g, 3 min, room temperature (RT). Remove supernatant. Resuspend cells in 620 µL RT 1X PBS.
  3. Aliquot 100 µL cells to the second set of labeled tubes that were set aside for cells.
  4. Spin cells at 600 x g for 3 min at room temperature (RT). Remove supernatant. Resuspend each cell pellet in 100 µL of the assigned Permeabilization Buffer (or Control) and incubate 10 minutes at RT.
  5. At the end of the incubation, examine each sample by Trypan Blue staining
    1. In a fresh 1.5 mL tube, mix 10 µL cells + 10 µL 0.4% Trypan blue. Load 10 µL onto a hemacytometer/cell counter slide.
    2. Count live (intact, Trypan negative) vs. dead (permeabilized, Trypan positive) cells. Select minimum Digitonin concentration that permeabilizes >95% of cells (example in Figure below).

Figure. 0.01% Digitonin is the minimum concentration that permeabilizes >95% K562 cells (black arrow). Cells were treated with CUT&RUN Wash Buffer containing various Digitonin concentrations and evaluated by Trypan Blue staining. Green cells (Trypan negative) are intact, whereas permeabilized/dead cells (Trypan positive) are red. Values (bottom right of each panel) indicate percent of dead/permeabilized cells.

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