Sack: Cell Plating for Electrophysiology

Sack and Yarov-Yarovoy Labs

Department of Physiology and Membrane Biology
University of California, Davis

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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