Adherent cells can be collected for CUTANA™ workflows by enzymatic digestion or phsyical scraping, as outlined below.
Enzymatic digestion
Collect adherent cells using mild enzymatic digestion to dislodge and disaggregate clumps into a monodispersion without cell damage. Optimize cell detachment and viability by adjusting enzyme concentration and duration according to cell type. Use either 0.05% trypsin or 0.25% trypsin (preferred), accutase, collagenase, or dispase for a maximum time of 30 minutes at 37oC. Avoid extensive enzymatic digestion and processing agents containing glucosaminidase, amidase, neuraminidase, or mannosidase to prevent interference with CUT&RUN.
Remove culture media and add enough enzyme solution to cover culture surface. Incubate at 37°C for the minimal time necessary to dislodge cells.
Add pre-warmed complete cell culture media at 5 times the volume of enzyme solution to inactivate enzyme. Transfer cells to a new tube and spin at 600 x g for 3 minutes to collect cells. Enzyme will be removed during subsequent wash steps.
Proceed to initial cell count and harvest cells for appropriate CUTANA assay.
Physical scraping
Scraping can lead to cells clumps. Throughout, repeatedly pipette cells to disassociate clumps.
Remove cell culture media and replace with enough fresh complete cell culture media to cover culture surface.
Gently scrape cells with cell scraper and collect into 15 mL tube.
Wash plate with a small volume of complete cell culture media to collect any remaining cells and add to tube. Examine culture surface under a microscope to ensure all cells are removed.
Thoroughly pipette to ensure monodispersion of cells.
Proceed to initial cell count and harvest cells for appropriate CUTANA assay.