Encyclopedia of Experiments: Cancer Research
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Transcript
Collect the sample from the OR and transport it on ice to the lab. Still inside the transported container, place the sample in a biological safety hood. Transfer the sample from a 50-milliliter conical tube onto a 60 millimeter by 60 millimeter Petri dish containing 10 milliliters of fresh ice-cold PBS.
Next, using a sterile razor blade, further cut the sample into 2 millimeter or smaller pieces. Then, treat DMEM with dispase II in a 15-milliliter conical tube. Transfer the minced tissues to the DMEM dispase II mixture tube. Then, incubate the sample at 5% carbon dioxide and 37 degrees Celsius for 30 minutes. Manually agitate the cell slurry every five minutes to ensure optimal digestion.
After incubation, transfer cell slurry through a 70-micrometer mesh cell strainer placed on top of a 50-milliliter conical tube. Gently apply pressure against the mesh using a syringe plunger. Discard any undissociated tissue remaining on top of the mesh and collect the obtained cell suspension in the 50-milliliter sterile conical tube.
Next, centrifuge the conical tube at 320 x g for seven minutes at 4 degrees Celsius. Discard the supernatant and resuspend the cell pellet in 10 milliliters of DMEM containing 10% FBS and 1% PS. Then, transfer the cell suspension to a Petri dish and incubate the suspension at 5% carbon dioxide and 37 degrees Celsius. Change the medium 24 hours after the initial plating to remove the cellular debris and the majority of the erythrocytes present in the culture.
Finally, change the medium every three days for the following two weeks, after which the cultures of primary EOC cells are ready for downstream applications.
