- Put coverslip with adherent cells in the sample chamber and wash with PBS. This involves gently spraying the PBS on the coverslip with a pippette, and gently rocking the sample chamber back and forth. This should only require about 0.5 mL of PBS.
- Aspirate PBS and fill chamber with 1 mL of 4% formaldehyde. Let sit for 15 minutes at room temperature (in cell prep box, covered with a chem wipe).
- There are variances in both time and temperature (some incubate rather than using room temperature), so this is something that could be varied if desired.
- Aspirate formaldehyde and wash twice with PBS. Aspirate PBS.
- For measurements, fill with HBSS at 37C as is typical with live cell measurements.
Tag: procedure
Cell culturing protocol
Thawing Frozen Cells
- Cells arrive deep-frozen.
- Add cells to 10 mL of growth media with 20% FBS.
- Incubate overnight or until confluent.
- Passage to 2 dishes with 10 mL of growth media with 10% FBS
- Continue to passage about twice a week.
Passaging (SCC7)
- Aspirate (remove) media.
- Add 1 mL of trypsin, coating the entire bottom of the dish.
- Swirl for 20 seconds, then aspirate.
- Add 1 mL trypsin.
- Incubate for 4 minutes.
- Use the pipette to “pressure wash” cells off of one dish with trypsin.
- Add the trypsin with the lifted cells to the second dish.
- Use pipette to wash cells off of the second dish with trypsin.
- Add (x) μL of trypsin with lifted cells to new dishes with 10 mL of growth media.
- (x) should be somewhere between 75 and 150, depending on how quickly you want the cells to reach confluence
Passaging (HeLa)
- Aspirate (remove) media.
- Add 10 mL of HBSS or PBS.
- Aspirate.
- Add 2-4 mL trypsin.
- Incubate for 3 minutes.
- Use the pipette to “pressure wash” cells off of one dish with trypsin.
- Add the trypsin with the lifted cells to the second dish.
- Use pipette to wash cells off of the second dish with trypsin.
- Add (x) μL of trypsin with lifted cells to new dishes with 10 mL of growth media.
- (x) should be somewhere between 75 and 150, depending on how quickly you want the cells to reach confluence
Cell Media/Solutions
Cell Media (EMT6)
- 500 mL BME (Basal Medium Eagle. We have always gotten this from the Foster lab)
- 2.5 mL Pen Strep
- 2 mL Fungizone
- 1 mL plasmocin
- 1 mL primocin
- 10% FBS – 55.5 mL (Fetal Bovine Serum)
*Note: 20% FBS media is also needed for when cells are first thawed.
*For SCC7 cell line, components are all the same except instead of BME base media, use RPMI media
Cell Media (HeLa)
- 500 mL RPMI 1640
- 10% FBS (50 mL)
- 5 mL Hepes Buffer
- 5 mL Pen Strep
*All Nada solutions are based on 500mL total final solution
Nada Solution #1 (basic cell measurement solution)
- 3.9447 g NaCl
- 0.2199 g KCl
- 0.9008 g D-glucose
- 6 mL Hepes
- 0.0832 g CaCl2
- 0.0571 g MgCl2
Nada Solution #2 (permeabilizing solution)
- 3.9447 g NaCl
- 0.2199 g KCl
- 0.9008 g D-glucose
- 6 mL Hepes
- 0.476 g MgCl2
- 20 μM Ionomycin
Nada Solution #3 (calcium shock solution)
- 3.9447g NaCl
- 0.2199g KCl
- 0.9008g D-glucose
- 6mL Hepes
- 0.0888g CaCl2
SUPERFASLIGAND (for 100 ng/10 uL concentration)
- 5 ug of SFL
- 500 uL of HeLa Media