Competition of Charged Molecules with Lipophilic Cations

Protocol

1. TF cells on coverslips with desired construct, and treat cells as required for the experiment.
   
2. Prepare 2x stocks of desired lipophilic compound (see table below for concentrations) in HEPES-buffered RPMI.
   
3. Transfer coverslip to imaging chamber; fill with 500μl of HEPES-buffered RPMI.
   
4. Pre-heat stage for at least 1hr prior to imaging. Turn off AC during imaging.
   
5. 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.
   
6. 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

Compound	[Stock]	[Working]	Vol. Stock -> xμl for 1mL of 2x solution
Dibucaine	1mM	100μM	100μl->900μl
Sphingosine	3.34mM	50μM	15μl->985μl
Squalamine	1mg/ml	80μg/ml	8μl->992μl
Ruthinium red*	1mM	50μM	25μl->975μl
Lanthanum Chloride**	1mM	100μM to 500μM	100μl->900μl to 500μl->500μl

• (*) 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.