Competition of Charged Molecules with Lipophilic Cations
From Heit Lab Wiki
- TF cells on coverslips with desired construct, and treat cells as required for the experiment.
- Prepare 2x stocks of desired lipophilic compound (see table below for concentrations) in HEPES-buffered RPMI.
- Transfer coverslip to imaging chamber; fill with 500μl of HEPES-buffered RPMI.
- Pre-heat stage for at least 1hr prior to imaging. Turn off AC during imaging.
- Identify 5-10 cells of interest, record position using point visiting. Once identified, let cells sit 5min, and then check focus. If good, go to next step. If focus drifts, allow another 5-10min equilibration period.
- Add 500μl of the 2x lipophilic compound. Quickly check focus, then begin a time-lapse with images taken every 20sec, for a period of 10min. Use the shortest exposure possible to limit photobleaching.
Table 1: Concentrations of lipophilic cations for charge competition
Vol. Stock -> xμl for
1mL of 2x solution
- (*) Ruthinium red function by altering intracellular Ca2+ levels. Due to its indirect method of action it should be avoided if possible.
- (**) Lanthanum chloride is a tri-valent cation. However, it also interferes with GABA receptors, and as such should only be used for cell-free assays.