• Created by Unknown User (amgarcia@mit.edu), last modified by Unknown User (lylaatta@mit.edu) on Jul 13, 2014 12:32
STEPS:
  1. MATERIALS: Ensure that trypsin is completely thawed, but PBS/versene and complete media are cold (do NOT place in warm water bath; take them straight from the fridge).
  2. Aspirate off media.
  3. Rinse wells with 500uL of PBS/versene. Aspirate.
  4. Add 200uL of trypsin/EDTA to each well. Incubate at room temperature for 90 seconds, then neutralize with 800uL of cold complete media.
  5. Triturate (pipette up and down) to disperse clumps. Transfer to cytometry tubes. Place tubes in ice (cells must be kept cold; in stasis).
    1. Number the wells and the corresponding tubes (the FACS machine deals with numbered samples better). Record the contents of the wells to which the numbers correspond.
  6. Count the cells from 2-3 tubes, using the hemacytometer (typically 2x10^6 per mL). Add PBS to dilute the cells to 1x10^6 per mL (typically, an additional 1mL is required).
  7. Run FACS.

 

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