Freezing and Thawing Cells in a 96-well Plate
Reagent | Vendor | Catalog Number |
---|---|---|
Corning™ Matrigel™ hESC-Qualified Matrix | Fisher Scientific | 354277 |
Falcon™ Polystyrene Microplates | Fisher Scientific | Various |
Corning™ Cell Culture PBS (1X) | Thermo Fisher / Life Technologies | MT21040CV |
DMEM-F12 | Thermo Fisher / Life Technologies | MT10092CV |
Essential 8 Medium | Thermo Fisher / Life Technologies | A1517001 |
Stemflex Medium | Thermo Fisher / Life Technologies | A3349401 |
mTesR Medium | Stemcell Technologies | 85850 |
Accutase | Innovative Cell Technologies | AT104 |
Cryostor CS10 | Stemcell Technologies | 07930 |
STEM-CELLBANKER® | Thermo Fisher / Life Technologies | 11897 |
Required Equipment / Consumables:
Freezing
- Add 50μl of Cryostor CS10 directly to the wells of a 96-well plate.
- Using a P200 pipet tip, gently scratch the bottom of the well to suspend the cells in the freezing media.
- AWrap the plate with parafilm, and store directly in -80°C in a freezing container.
Thawing Directly to 24-well Plate:
- Remove plate from -80°C and thaw for about 5 minutes in a 37°C incubator.
- Add 200 μl of basal media directly to the 96-well plate and pipet up and down gently.
- Add 250 μl of basal media to 1 well of a Matrigel-coated 24-well plate.
- Add the resuspended cells into the well of a Matrigel-coated 24-well plate (total volume: 500 μl). Incubate at 37°C.
- Feed cells every day until confluent.
Thawing and Centrifuging:
- Remove plate from -80°C and thaw for about 5 minutes in a 37°C incubator.
- Add 200 μl of basal media directly to the 96-well plate and pipet up and down gently.
- Transfer resuspended cells to a 1.5mL eppendorf tube.
- Add 250 μl of basal media to resuspended cells.
- Spin at 300g for 3 minutes.
- Aspirate the supernatant, leaving a trace amount to prevent the pellet from drying.
- Resuspend cell pellet in 250 μl of basal media.
- Add the resuspended cells into a well of a Matrigel-coated 96-well plate. Incubate at 37°C.
- Feed cells every day until confluent.
Which way is the best way?
Thawing directly to a 24-well plate is a much faster way, but thawing and centrifuging the cells yields a higher cell survival rate.