14. Splitting cells procedure - dfierros/cohen_lab GitHub Wiki

Warm up media in water bath

• When taking out of water bath, wipe off water before spraying down.

Remove dish to be split from incubator and place in hood Aspirate off media. Wash with PBS (7 mL minimum) by spraying on side of dish then swirl around

• Enough to get good coating but not dry out
• Note, don’t always need to do wash out step if just doing quick split Aspirate again. Add trypsin (3 mL for 60 mm plate, 5 mL for 100 mm plate, 7 mL for 150 mm plate--see chart) Incubate for 7-10 minutes.

This incubation is the best time to do stuff, like:

Label dishes you are splitting into (this step can also be done during centrifuge)

• Add media to dishes ** 3 mL (can do 4 if going for a Friday-Monday plate) for 60 mm plate ** 20 mL for big plate ** 10 mL for 100 mm plate

Get plate from incubator. Check cells under microscope.

• Should be separated
• Swirl or tap to separate (tapping causes mitotic shake off)

Add media to neutralize trypsin (~1 to 1 ratio to trypsin) Pipet out of dish and put in a tube (no need to swirl) Centrifuge (0.3*1000 RCF, 3 minutes)

• Make sure evenly balanced on either side with spare tubes

This is the most dangerous step. Bring warm media into the hood if not already there

Aspirate the liquid from the tube, but don’t suck up the pellet.

Pipette media (around 950 microliters - assuming ~50 microliters pellet) into tube. Triturate. If you are going for 2e6 cells/mL or some density, go with 450 uL or less if your plate isn’t super dense

• Don’t create froth/bubbles.
• Keep tip fully submerged.
• Check pipetted volume.
• Only push down to first stop
• Pipette into desired dish.
• Remember to retriturate each time
• Distribute droplets evenly
• Drop more close to edges than center
• Swirl in figure-8.

Check on scope to make sure there’s a reasonable amount of cells