We have difficulties with a neuropharmacological assay.
We use HEK-293 cells stably transfected with the human receptor TRPV1. We monitor intracellular calcium using Fura2.
We stimulate these cells (coated on 96-well plate) with capsaicin (10-100 µM) and measure the fluorescence with a FLEX station. We are unable to observe an increase of intra Ca2+.
Does anyone have any advice on possible experimental precautions to be taken in this assay?
Thank you very much.
Hi Cesar, check the pH of your extracellular solution, freshly prepared HBSS + 0.1% BSA + 10 mM HEPES, pH 7.4, in warm solution for experiments performed at 37C. Also try capsaicin at a lower concentration of 1 micro Molar. Can use DMSO to make capsaicin solution at 100 miliMolar concentration (wear a mask and gloves) (store stock solution aliquots at -20C), and prepare intermediate dilutions 1000x with ethanol. Cells tend to respond after at least 48 hours after plating. Also, need to use geneticin to select TRPV1 expressing cells.
Check this link
Ratiometric Ca²⁺ measurements using the FlexStation(®)Scanning Fluorometer. Marshall IC, Boyfield I, McNulty S. Methods Mol Biol. 2013;937:95-101. doi: 10.1007/978-1-62703-086-1_4. PMID: 23007580
Our Flex station has a UV detector and we do ratiometric measurements. This, at least, works quite well.
You should also use a positive control by stimulating an endogenous receptor in the HEK293 cells.
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