At the Faber Lab with Janette
tryPLE is ok to use for organoids pre-warm plates in incubator (15 min ok) In a 24-well plate, 3 droplets of matrigel per well, ~10ul of cells (in media) per droplet
matrigel can only be changed when passing cells
keep small ice bucket in flow cabinet for matrigel - matrigel must be cold to break
When handling cells, ALWAYS coat tip in FCS or you will lose all your cells in the pipette tip - don't learn the hard way!
Remove media from cells, observe droplets (cells grow only on the matrigel)
Take 500ul media (ice cold), use to break droplets - pipette up and down with some force; eject into cold media, repeat 1x.
Remember to coat pipette tip with FCS!
Spin cells 5 minutes, 4C, 500 RCF
Fill wells at the outer border of plate w/H2O or PBS for humidity; remove this when you split the cells, replace when you are done
After the spin, aspirate most media, leave ~500ul. Keep the tube cold on ice. Set P200 to 180ul. Coat pipette tip in FCS. Pipette up and down at least 50x by holding pipette 90 degrees with a rapid press/release motion (forceful but do not cause the tube to overflow). Check for uniformity. If matrigel too thick keep ice cold to break, then add RT or cold media (NOT HEATED) and spin again (same spin settings).
Pipette off as much media as possible w/P200 (ok to vacuum first mLs). At this step it is ok to not coat pipette tip with FCS because you don't have cells). Leave ~60ul - amount left is determined by how many new wells seed (30ul/well final)
Use P20 to make droplets; set to 10ul. Avoid making bubbles in droplets
Matrigel aliquot on ice, pipette up and down, mix with cells but keep on ice. 70% matrigel final; a bit extra is better than not enough. Can add 4th droplet if extra, or add more to existing droplet
After adding matrigel/cells put plates without media upside-down in incubator to trick the cells into sticking in the matrigel, they will prefer to stick to the plate otherwise. Leave 10 minutes.
Add 500ul organoid media/well. Fill outer wells with H2O or PBS.
Media Changes Remove media with pipette. Add new media on the side of the well, do not touch matrigel!
One well = one cryotube; thaw into one well