How should cells be dissociated from RetroNectin-coated plates?
How should cells be dissociated from RetroNectin-coated plates?
For strongly adherent cells, like fibroblasts, use trypsin-EDTA (without Ca2+ and Mg2+) to dissociate cells.
For weakly adherent (or suspension) cells, use a 0.02% EDTA/PBS solution following the protocol below. Use trypsin to remove these cells only if the EDTA/PBS treatment is unsuccessful; trypsin may damage these cells.
1. Following transfection, transfer the supernatant from the plate to a centrifuge tube.
2. Wash the plate with PBS to recover non-adherent cells.
3. Dissociate adherent cells from the plate using Cell Dissociation Buffer (Thermo Fisher Scientific; enzyme free, PBS-based) following the manufacturer's instructions.
4. Combine all obtained cells in one centrifuge tube, and centrifuge to recover the cell fraction.
5. Wash cells with HBSS/HEPES twice, collect by centrifugation, and suspend cells in HBSS/HEPES for further use.