Care and Handling of Feeder Layer Cells
STO/SNL cells are a derivative of the standard STO cell line. They are transformed with a LIF producing plasmid (low-level). They grow quite well, and look slightly more vacoulated than regular STO cells. This line was made available to use from the Bradfield lab.
STO Cells
Media
High glucose DMEM (-pyruvate,-glutamine)
Newborn calf serum (heat inactivated 56°C for 30 min)
1X l-glutamine (add fresh every 2 weeks)
1X penicillin/streptomycin
Add all components to DMEM, sterile filter if desired
Thaw frozen vial of cells
Thaw rapidly at 37°C, rinse outside of vial with 70% ethanol
Use a lml pipet to transfer cells to a 15ml centrifuge tube, add 5ml STO/SNL media
Spin down cells for 2 minutes at 1000 rpm
Aspirate off media and resuspend cell pellet in fresh media, plate
Growing cells
Grow cells in 10 cm tissue culture dishes
Change media every 2 days
Split cells 1:10 as soon as they become confluent (usually ~ 4 days)
Splitting cells
Aspirate all media from plate and add ~5 ml. incomplete PBS. aspirate. Repeat 2X. Add 3ml. trypsin-EDTA.
Put plate in 37°C incubator for 8-10 minutes
Tap plate to check that cells are loosened
Add T-E solution to an equal volume of media in a centrifuge tube.
Pipette back and forth a few times to insure a single-cell suspension. Spin 2 min at 2000 rpm.
Aspirate media, resuspend cell pellet in fresh media and plate. Usually cells are plated 1:8.
STO/SNL Cells
Media
High glucose DMEM (-pyruvate,-glutamine)
Newborn calf serum (heat inactivated 56°C for 30 min)
1X l-glutamine (add fresh every 2 weeks)
1X penicillin/streptomycin
Add all components to DMEM, sterile filter if desired
Thaw frozen vial of cells
Thaw rapidly at 37°C, rinse outside of vial with 70% ethanol
Use a lml pipet to transfer cells to a 15ml centrifuge tube, add 5ml STO/SNL media
Spin down cells for 2 minutes at 1000 rpm
Aspirate off media and resuspend cell pellet in fresh media, plate
Growing STO/SNL
Grow cells in 10 cm tissue culture dishes
Change media every 2 days
Split cells 1:10 as soon as they become confluent (usually ~ 4 days)
Splitting STO/SNL cells
Aspirate all media from plate and add ~2ml 1X trypsin, swirl so all cells are covered
Put plate in 37°C incubator for 5 minutes
Tap plate to check that cells are loosened
Add 3ml media to plate, pipet up and down several times to achieve a single-cell suspension
Transfer cells to a 15ml centrifuge tube, spin 2 min at 1000 rpm
Aspirate media, resuspend cell pellet in fresh media and plate
Freezing STO/SNL cells
Trypsinize either regular or MitoC-treated STO/SNL cells as aboveAfter centrifuging resuspend cells in freezing media (STO/SNL media with 20% NCS and 10% DMSO)Volume should be approximately lml per plate of cells, aliquot to cryovials adding lml per vialFreeze vials immediately at -20°C for ~2 hr, then at -80°C overnight, transfer to liquid N2 the next morning.
Growth-Arrest for Feeder Layers
Split cells as described above. However, instead of plating, transfer the cells (in media) to a sterile centrfuge tube. Parafilm the tube, and place the cells in a small ice-bucket. Bring the cells down to the gammacell (you MUST be certified to use the gammacell. See Rex for information about certification). Place the cells in the sample cavity (don’t forget to sign in) and irradiate for 40 minutes. Wipe off the tube with 70% ethanol, and place the tube back in the hood. Plate the cells, using 1 full plate of cells for 1 plate of feeders. One 10cm2 plate makes enogh feeder cells for another 10cm2 plate, 2 24-well plates, or 2 96-well plates. Check the cells and change media (to ES cell media) the next day. It is not unusual to see some death, but normally the cells spread out enough that this does not cause problems. You want the cells to cover the entire plate, confluency is preffered. You do not wish plated ES cells to be exposed to open surfaces on the plate (although they generally only stick to the feeder cells anyway).
Plating MitoC treated feeder cells
A confluent 10 cm plate of STO/SNL cells will make: 2 10 cm plates, 2 6-well plates, 1 24-well of feedersAdd the appropriate MitoC-treated cells to gelatinized wells in STO/SNL mediaAllow 12 hours for cells to attach before using feeders. If after 12 hours feeder cells appear too thin more may be added.
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