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Subculturing Cells - Cell Culture

Cell Culture

Cell Subculture Protocol


  • Get DMEM media 4C; Trypsin-20C; BSA-20C;
  • Put in 36mL of DMEM into conical tube plastic
  • Add 4mL of BSA media to 36mL DMEM;
  • Get syringe and filter;
  • Suck up media with syringe.  Add filter. 
  • Push out media through filter on syringe.
  • Repeat twice to get all of volume. 
  • Put into new plastic tube conical. 
  • Remove old media by dumping into beaker; DO NOT USE ASPIRATOR (get contamination!!)
  • Add 3mL typsin to culture.  Trypsin with pull cells off the culture container.
  • Put container into incubator.  Wait 2 min; Take out;  look into microscope. 
  • Try to get less clumps.  More individual cells.
  • Remove most of trypsin by dumping into beaker.
  • Put  back into incubator. Check frequently.
  • Slap container on sides to check bottom for less clumps; look into microscope.
  • When see many individual cells floating along; Great!
  • Add media to larger container; 1:5/ 1:3 dilution (3-5 containers larger).
  • Add 10mL to (T75)large container, 5mL to (T25)small container.
  • Suck out Most (5mL of cells from T25). Add to T75.
  • Leave some cells in small container.  Add media to small container.  Check in microscope for single cells;  Incubate in 37C incubator. 
  • See other Cell-protocols at our Cell Biology and Cell Culture Protocol Directory.


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