Cell Culture Protocol

• Transfer 2x10^5 viable hybridoma cells into 10mL warm, complete culture media into a T25 culture flask. Incubate, in a humidified incubator, at 5% CO2 and 37C.

  
  

  
  
• Check the flask daily under the microscope to assess growth and general health of the cells

  
  

  
  
• On day 4 after culturing, take a viability count of the cells using trypan blue. Generally, the cell numbers will have increased ten-fold

  
  

  
  
• Make a 1:10 dilution of the cells into a new flask with fresh media and incubate.

  
  

  
  
• The cells which were not transferred to a new flask can be expanded into larger flasks, at the same dilution, in preparation of experimental use.

  
  

  
  
• Estimates of cell yields can easily be calculated and experimental protocols designed

  
  

  
  
• For laboratory reference purposes, generate a growth curve from day 0 through the day on which few than 50% of the cells are viable