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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.
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