Calcium assay: Buffer gives increase in signal, similar to agonist - (Oct/29/2010 )
I am trying to measure intracellular calcium increase using Fluo-4 NW kit from Molecular probes. Briefly, I take off media from wells in 96well plate and wash the cells with PBS. I incubate with dye 30min at 37degC followed by 30min at RT. I then remove the dye and add my assay buffer (I use physiological salt solution).
I use Varioskan plate reader with dispenser for the experiment. When I added my assay buffer I got a spike for increased fluorescence and it stayed for about 100sec and it gradually came down (just like an agonist response curve).
Could anybody please help me get around this?
My buffer has 1mM calcium as I am looking at GPCR activation. I also use 10uM Ionomycin as my positive control.
I have tried dispensing at the lowest speed possible with as little volume as 10ul. Still it gives a response. I cannot do anything before I can sort this out.
I'm a little confused by your post. are you saying that without any other compound just simply adding NW buffer is causing a steady increase in Calcium flux? are you getting the machine to load the dye?
For some reason I am thinking that the osmolarity of the two buffers are different and you are seeing your cells 'readjusting' to their new conditions. Cant you just run the assay 5' after you load the NW buffer?
On the other hand if you're saying that adding a vehicle causes this spike i'm guessing your cells are responding to your vehicle (DMSO, ethanol, etc) and that you should reduce the concentration of your solvent.
Hope that helps
Hi sorry Mark was off for a long time! I found that my compounds are sticking to the tube! and the machine was pretty cranky as well! kept on missing wells to dispense in! Thanks for your reply though