Basic cell culture instructions

Troubleshooting Low Hemacytometer Counts

1. Trypsinization not complete:

   1. Trypsin is ineffective

      • too cold, be sure to warm sufficiently

      • self digested or expired check date, don't warm too long

      • too much serum left on plate rinse plate thoroughly with PBS

   2. Trypsinization technique

      • Trypsin doesn't coat plate, completely add full 2 mls, lay flask down, count to 10, then remove

      • trypsin left on plate too long and then aspirated...cells removed along with trypsin

      • not left long enough in incubator depends on cell line 3T3-L1 can go 1-5 minutes

      • flask may need to be tapped or slapped to facilitate cell removal
        (this varies by cell line, but ok for 3T3s)

2. Resuspension technique

   • too much media added more media results in low cell/ml, but overall cells on plate should remain the same

   • cells not sprayed off surface properly

   • media and cells not pipetted (gently) up and down 3-4 times to break up clumps

   • too long of time before retrieving sample from flask (cells may settle)
     
     After mixing with trypan, don't wait too long before loading hemacytometer.  Get hemacytometer ready while trypsinizing cells in incubator

3. Stubborn cells

   • cells left on plate a long time (>4 days) will be more difficult to remove

   • very confluent plate will require more aggressive trypsinization because trypsin cannot recach plate surface effectively