Sack: Cell Harvesting for Electrophysiology
Cell Plating for Electrophysiology
Jon Sack Feb 3, 2011
Ken Eum April 12, 2011 (Edit)
Notes: Steps should be done (not necessary if cell stock is only being used for plating cells for electrophysiology) inside of a Biological Safety Cabinet in Sterile conditions.
Procedures (in 35mm dish):
- Check under microscope to make sure that cells are less than 80% confluent
- Carefully remove (aspirate) the solution from the T-25 flask containing the cells
- Rinse with DPBS (Divalent free Dulbecco’s Phosphate Buffered Saline) ~ 5 mL
- Remove (aspirate) the DPBS solution
- Add 1 mL 0.05% Trypsin EDTA
- Place in 37° C incubator until cells detach from the dish ~ 5 min
- Check under the microscope to make sure cells have detached. If the cells are not yet detached, lightly tap the dish against the table.
- Pipet up and down 5-10 times in the Biological Safety Cabinet
- Check under the microscope to make sure the cells are dispersed into single cells.
- Add 2 mL of Cell Media with selection agents into new 35mm dishes
- Add 1 drop of the trypsinized cells into each 35mm dish with the Cell Media
- Swirl the dish or pipet up and down
- If using Kv2.1 T-Rex CHO cells, add tetracycline (1µg/mL) for 1 hour. After 1 hour, replace with fresh media or the external solution for electrophysiology recordings.
- Label the Dish: Name, Date, Cell Line, Passage #, Ratio
- Cell Media: Ham’s – F12 from Gibco, 10% FBS, 1% Penicillin/Streptomycin
To make add 50 mL Media + 50 mL FBS
- Cell Media with Selection Agents (Zeocin and Blasticidin)
F12 from Gibco, 10% FBS, 1% Penicillin/Streptomycin, 1/10,000 Blasticidin, 1/4,000 Zeocin