View source for Competition of Charged Molecules with Lipophilic Cations

= Protocol =

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

===== Table 1: Concentrations of lipophilic cations for charge competition<br> =====

{| cellspacing="1" cellpadding="1" border="1" style="width: 534px; height: 135px;"
|-
| '''Compound'''<br>
| '''[Stock]'''<br>
| '''[Working]'''<br>
| 
'''Vol. Stock -&gt; xμl for<br>'''

'''1mL of 2x solution'''

|-
| Dibucaine<br>
| 1mM<br>
| 100μM<br>
| 100μl-&gt;900μl<br>
|-
| Sphingosine<br>
| 3.34mM<br>
| 50μM<br>
| 15μl-&gt;985μl<br>
|-
| Squalamine<br>
| 1mg/ml<br>
| 80μg/ml<br>
| 8μl-&gt;992μl<br>
|-
| Ruthinium red*<br>
| 1mM<br>
| 50μM<br>
| 25μl-&gt;975μl<br>
|-
| Lanthanum Chloride**<br>
| 1mM<br>
| 
100μM to

500μM<br>

| 
100μl-&gt;900μl to<br>

500μl-&gt;500μl<br>

|}

<br>

*(*) 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.<br> <br>