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Comparative Physiology @ Guelph

Culturing Keratinocytes Protocol & Tips

General Tips:

  • Turn the UV light on in the sterile hood for 20 min before use
  • Ethanol down your sterile gloves and sterile hood before use
  • Ensure your pipette tips have been autocleaved and left in the sterile hood
  • Make sure you are using Tissue Culture Plates and not just standard petri plates or else your cells won’t attach/grow
  • To calibrate the incubator, use the Fyrite Calibrator found in Dr. Mosser’s lab (The liquid must be changed yearly for accurate measurements)

Thawing Cells:

  • Warm the frozen vial of cells in a 37C bath for ~2 min until they are just barely thawed
  • (Tip: warm the cells until you can invert the vial and see the liquid sample move from the bottom of the vial to the top. As soon as you can do this your cells are ready)
  • In the sterile hood, pipet the cells gently and slowly from the vial onto the culture plate
  • Add 1mL of RM- media into the vial to grab the rest of the cells & pipet them onto the culture plate as well
  • Slowly add the rest of the RM- media to the culture plate so that the cells aren’t diluted too fast (~1-2min)
  • (Tip: Add 9mL to make up 10mL total RM- when using a large culture plate 100mm X 20mm)
  • Put the culture plates in the incubator (5% CO2, 37C) and check on them under the microscope in 24hrs, look for attachment of the cells to the plate. If attachment has occurred, change the media from RM- to RM+ media — otherwise wait another 24hrs and check again for attachment

Maintaining Cells:

  • On average, every 48 hrs the media should be changed to ensure that the cells survive
  • (Tip: RM- and RM+ media is Red; if it ever turns deep yellow you’ve waited too long to change the media and you must change it immediately)
  • Aspirate the RM+ media off and apply the new warmed (37C) RM+ media to the cells + place back into incubator
  • (Tip: 10mL of RM+ media is the right amount for a large culture plate 100mm x 20mm)

Splitting Cells: (for a large plate 100mm x 20mm)

  • Aspirate off the media and add 5mL of PBS solution (pH 7.4) for 5 seconds (This will wash the cells off)
  • Aspirate off the PBS solution and add 5mL of PBS:EDTA solution (pH 7.4) for 2 min (PBS:EDTA will help the cells lift off the plate like trypsin, allow it to sit for 2min as Trypsin lifting the cells alone can be harsh)
  • Aspirate off the PBS:EDTA solution and add 1mL of Trypsin, put the plate in the incubator for 5-10min (This will lift the cells, however it can be harsh so only leave it on as long as needed to just lift the cells if left too long the cells will die, check under the microscope to see the cells lift)
  • As soon as the cells lift, add 5mL of RM- media to the plate with trypsin (6mL total) (The FBS in the media will inactivate the trypsin preventing cell damage)
  • Split the cells in a 1:5 or 1:10 ratio (Tip: 1mL of this solution to 5 large culture plates is a good split)
  • Add RM- media to the new culture plates to make up a total of 10mL (Addition of 9mL to the 1mL taken from the trypsin plate)
  • Return the plates to the incubator and check in 6-24hrs if the cells are attached to the plate -> changed the media to RM+