Flow Cytometry Sample Preparation Optimization
Sample preparation and cell quality tips
- Whenever possible, use freshly isolated cells rather than frozen and thawed cells.
- To increase viability of thawed cells, perform the initial dilution of the thawed cells at a high serum concentration (90% FBS in culture medium).
- When isolating populations rich in adherent cells, use non-tissue culture treated plastic dishes and tubes.
- When isolating cells from complex tissues, it is better to perform the steps on ice (except for the steps involving digestion enzymes) to prevent phagocytosis and cell lysis.
- To prepare homogenous single-cell suspensions, gently pipette the cells gently as opposed to vortexing in order to avoid cell disruption.
- If the downstream procedure is live cell sorting, it is recommended that cells are counted after each step to ascertain viability.
- Minimize dead cells in the final suspension by removing clumps and other debris by sieving through nylon mesh.
Keywords: FACS protocol, flow cytometry cell preparation, adherent cell sample