1. IMR90 (Human fibroblasts)
2. PGP1 (Hu iPSCs)
For culturing Hu iPSC (PGP1) I use Wicell Feeder–Independent Pluripotent Stem Cell Protocols mTeSR™1 Medium.
3. PGP1 primary fibroblasts (PGP1F)
Freezing IMR90 cells (Caroline)
- Make freezing media 90% FBS and 10% DMSO. For example, 25 ml FBS, 2.78 ml DMSO. Stable in 4c for about a week.
- Trypsinize 1 or more plates of confluent cells. Collect the cells you want to freeze by centrifugation.
- Resuspend in freezing media drop-wise at room temp. Each 10 ml plate is good for 2-5 tubes.
- Add 1 ml to each cryovial. BE CAREFUL NOT TO UNDERTIGHTEN OR OVERTIGHTEN VIALS…this will allow liquid N2 vapor to get into the vial and will make it explode upon thawing.
- Place the cryovials in the “Mr. Frosty” cell freezing container. Make sure the container is filled with isopropanol to the fill line. Caroline or Sonny should know what/where this piece of equipment is…it gradually freezes the cells.
- Put the “Mr. Frosty” in the -80C freezer for 2-4 days.
- If you are freezing this cell line for the first time thaw a tube to make sure you froze it fine (see if it grows fine).
- After the 2-4 days, transfer the cryovials to the liquid N2 tank for long term storage
Thawing IMR90 cells
- Pre-heat media and PBS in 37c bath.
- Prepare a box with ice for the tube. Use dry ice.
- Take a tube from the liquid nitrogen tank and put in box with ice. Use eye protection and open tank and tube carefully. Empty liquid nitrogen into tank. When done with dry ice, open box in an open environment.
- Prepare 9 ml media in 15 ml tube.
- Hold 1 ml tube in 37c bath from the top for 90 sec to 3 minutes. Stop when only a small chunk hasn’t thawed yet. Hold the tube neck above water.
- Pipette up and down until it dissolves. Transfer to the 15 ml tube with media.
- Centrifuge at 300g (rcf) for 5 minutes at RT.
- Aspirate the media of the tube and suspend cells in new media (~8 ml).
- Transfer cells into plate. Put in 37c.
- Change media in next day.
- Passage/expand when they show 85% confluent.
- Remove media.
- Wash with PBS X2.
- Add 1 ml Trypsin.
- If plate is confluent dilute 1/5 (add 4 ml media to dish with 1 ml trypsin) and transfer to flacon tube.
- Mix 100 μl of cells with 900 μl PBS in an eppendorf.
- Take Millipore cell counter and 60 μm chambers.
Turn on the Scepter™ cytometer by pressing the control button on the back of the instrument and wait for on-screen instructions to appear.
- When prompted, attach a sensor to the end of the Scepter™ unit with the electrode-sensing panel facing toward the front of the instrument, and you’ll see detailed instructions for each step of the counting process.
- Pipette once to draw sample into the sensor. 50 uL of your cell suspension is drawn into the microfabricated, precision-engineered channel embedded in the sensor. The cell-sensing zone detects each cell drawn into the sensor and thus cell concentration is calculated.
The sensing zone also measures cell sizes and cell volumes with sub-micron and sub-picoliter resolution, enabling the Scepter™ cytometer to display a histogram distribution of cell size or cell volume.
- Seed in a concentration of ~106 cells/ml, 200 μl is enough for MatTek dishes.
- Leave in 37c ~4 hours.