Admin: Created page with "= 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..."

2021-02-01T19:56:10Z

Created page with "= 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..."

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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 =====

{| cellspacing="1" cellpadding="1" border="1" style="width: 534px; height: 135px;"
|-
| '''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.

Competition of Charged Molecules with Lipophilic Cations - Revision history

Admin: Created page with "= 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..."

Admin: Created page with "= 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..."