Competition of Charged Molecules with Lipophilic Cations
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Protocol
- 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
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.