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
- 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.
- Prepare the other four Cell Permeabilization Buffers by serial dilution (see Table). Vortex each buffer to mix and place on ice.
- 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
- 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).
- Spin 600 x g, 3 min, room temperature (RT). Remove supernatant. Resuspend cells in 620 µL RT 1X PBS.
- Aliquot 100 µL cells to the second set of labeled tubes that were set aside for cells.
- 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.
- At the end of the incubation, examine each sample by Trypan Blue staining.
- 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.
- 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.
