Membrane depolarization – luciferase assays

12/6/04

Membrane depolarization solution:

For 500mL, to 450mL ddH2O add

170mM KCl 6.33g KCl

10mM Hepes pH 7.4 1.19g Hepes

1mM MgCl2 101mg MgCl2

2mM CaCl2 147mg CaCl2

pH to 7.4 with NaOH and make to 500mL

Transfect neurons on DIV3-4 in culture

Best inductions in embryonic neurons grown in serum-containing medium

Also can use postnatal neurons in Neurobasal-B27, may need to block overnight with TTX and APV before use to get background down.

Two days after transfection, stimulate neurons by adding 0.45mL depolarization solution (prewarmed) to each 1mL medium.

After 5-8 hours harvest cells for dual luciferase measurement (Promega), 100uL lysis per well of 12 well plate, use 20uL to measure.