Standard sample prep workflow
High-quality sample prep is essential for CUT&RUN experimental success. This guide is for fresh, native (i.e. unfixed, not frozen) suspension cell culture. For alternative sample types (i.e. adherent cultures, tissues, cross-linking, frozen samples), find guidance in this section.
- Count starting cells and confirm cellular integrity, morphology, and viability. It is important that cells have good starting viability, prior to being resuspended in CUT&RUN Wash Buffer. For K652 cells, we aim for >90% viability.
- Harvest 500,000 live cells per reaction plus 10-20% excess. Spin at 600 x g for 3 min at room temperature (RT). Remove supernatant by pipetting, leaving a small amount of liquid on the pellet to avoid sample loss.
- Resuspend cells in 100 µL per reaction RT Wash Buffer by gentle yet thorough pipetting. Spin at 600 x g for 3 min at RT. Pipette to remove supernatant.
- Repeat Step 3 one time.
- Resuspend cells in 105 µL per reaction RT Wash Buffer by gentle yet thorough pipetting.
- Count and examine integrity of prepared cells by Trypan Blue staining.
- Add 100 µL cells to 10 µL ConA beads in 8-strip tubes. Gently vortex to mix and quick spin in a mini-centrifuge to collect slurry (beads should not settle).
- Incubate bead-cell slurry for 10 min at RT to adsorb cells to beads.
- Place tubes on a 8-strip tube magnetic rack and allow slurry to clear.
- If bead binding was successful, the supernatant should not contain cells. Save 10 µL [unbound fraction] to confirm (see Figure 1).
- Discard remaining supernatant and move quickly to the next step. Do not allow beads to dry out.
- Remove tubes from the magnet. Immediately add 50 µL cold Antibody Buffer to each reaction and pipette to resuspend.
- Transfer 10 µL of bead-cell slurry to a new 1.5 mL tube [bead fraction].
- Examine bead-cell slurry [bead fraction] and supernatant [unbound fraction] using Trypan Blue staining to confirm ConA bead binding. As described in Step 10: supernatant should contain few to no cells, bead-cell slurry should contain cells bound to beads (Figure).
Figure 1. (A) Supernatant [unbound fraction] shows little to no material leftover after ConA Bead conjugation. (B) Representative bead-cell slurry [bead fraction] image showing nuclei (blue) successfully conjugated to activated ConA Beads (brown specs). Note: ConA Bead-bound cells will also appear Trypan Blue positive due to the presence of Digitonin in the Antibody Buffer.
