Protocols

Cell Splitting Protocol

1. Turn on 37° water bath, Open TC hood, turn on light and vent, and spray counter with 70% ethanol. Periodically spray gloves with ethanol throughout the procedure.

2. In the hood, pipette 125mL of DMEM++ into a separate sterile container. Warm the DMEM++, PBS, and Trypsin containers in the bath.

3. After warming up for 20 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 vacuum line. Use the glass pipette to drain the medium from a corner of the old flask. With the 5mL pipette, rinse old flask with 4mL of PBS. Drain with vacuum.

7. Pipette 2mL of Trypsin into old flask and close the cap. Set timer for 4 minutes and put in incubator. Use the microscope to verify cell detachment. If preparing samples for imaging, incubate for another 4 minutes to detach more cells.

8. Spray flask 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. Using the same pipette, transfer less than 1mL of cells into an 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 sum the two counts. Multiply by 1000 to calculate the # of cells/mL.

14. Calculate the volume required for the desired number of reseeded cells, and pipette that volume into new T75 flask. To plate the MatTeks, first add more DMEM++ into the flask of cells such that there are approximately 5x10$ ^4 $ in 0.5 mL. Pipette 0.5 mL into each MatTek. Close the caps, 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 extra DMEM++ and PBS in the fridge. Trysin goes in the -20°C freezer.
• Wipe the counter and close the hood. Clean hemacytometer. 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.

3.7% Formaldehyde

• One 16% ampule
• 3.3 mL 1xPBS

Store in dark at room temperature.

0.1% Triton X-100

• 0.05 g Triton X-100
• 50 mL PBS

Store at room temperature.

1% BSA

• 0.5 g of BSA
• 50 mL PBS

Store at 4°C.

10uM cytochalasin D

• 50uL of 2mM cytoD
• 10mL PBS

Store at -20°C.

Alexa dye solution

• 7uL of dye in methanol
• 200uL of PBS

Microsphere Cleaning

1. Pipet 20uL of bead solution and 80uL of molecular grade water into centrifuge tube.
2. Centrifuge the microspheres at 1300 G for 10 minutes to clear the supernatant.
3. Remove and discard supernatant. Resuspend the microspheres in 160uL of water.
4. Sonicate for 10 minutes to break up aggregates.
5. Centrifuge at 1300g for 10 minutes.
6. Pipet out supernatant and bring into TC hood. Allocate 10mL of DMEM++ into Falcon tube. Transfer 0.3mL of beads into Falcon tube.