Protocols
Cell Splitting Protocol
1. Turn on 37° water bath and put in DMEM++, PBS, and Trypsin containers.
2. Meanwhile, open TC hood, turn on light, and spray gloves and counter with 70% ethanol. Clean the following items with ethanol before placing in hood:
- T75 flask
- MatTek dishes (if plating)
- vacuum tube
- electric pipette
- metal container of glass pipettes
- Plastic pipettes: 1x 25mL, 1x 10mL, 1x 5mL, 2x 2mL
- 1.5mL eppendorf tube
3. After warming up for 20-30 minutes, take DMEM++, PBS, and Trypsin out of bath. Dry them, wipe with ethanol, and bring into hood.
4. Add 25mL of DMEM++ into the new T75 flask. Add 2mL into each MatTek dish. (if plating)
5. Fetch flask of cells from incubator and make sure they look ok under the inverted microscope. Spray with ethanol and bring into hood.
6. Load a glass pipette into narrow end of vacuum tube. Attach wide end of tube to the hood. Tilt the old flask, and use the glass pipette to drain the medium.
7. With the 5mL pipette, rinse old flask with 4mL of PBS. Drain with vacuum.
8. Pipette 2mL of trypsin into old flask and close the cap. Set timer for 4 minutes and put in incubator.
9. Use the microscope to verify cell detachment. If preparing samples for imaging, incubate for another 4 minutes to detach more cells.
10. Spray with ethanol and bring into hood. Pipette 8 mL of DMEM++ into flask of trypsinized cells. Make sure to spread the medium and wash the cells from the walls by pipetting up and down.
11. Using the same pipette, transfer ~1mL of cells into eppendorf tube. Take eppendorf out of hood.
12. Transfer 90uL of cells and 10uL of Trypan blue dye into new eppendorf. Load each chamber of the hemacytometer with ~15uL of the cell-dye mixture.
13. There should be 9 large squares in the FOV. Count the number of cells in the 4 large corner squares and the large center square. Repeat with second chamber and average the two counts. Multiply by 1000 to calculate the # of cells/mL.
14. Calculate the volume required for 5x10$ ^4 $ cells, and pipette that volume into new T75 flask. Pipette Close the cap, label, and place in incubator.
15. Cleanup:
- Vacuum remainder of cells. Drain 50mL Falcon tube of bleach to wash the system. Disconnect vacuum tubes.
- Put DMEM++ and PBS in the fridge. Trysin goes in the -20°C freezer.
- Wipe the counter and close the hood. Toss eppendorf, old flasks, and gloves in the biowaste container.
Preparing Solutions
DMEM reconstitution
For 1 L of DMEM++
- In the hood, combine
- 1L DMEM
- 100mL FBS
- 10mL Pen/Strep
- Label as DMEM++ and store at 4°C.
