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

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