[Cytometry] Calcium flux + surface markers
helen.ferry at ndm.ox.ac.uk
Thu May 19 12:16:00 EDT 2011
I have a user who I have been trying to help establish a new calcium flux assay for a subset of human T cells. Originally they were just using Fluo-3/FuraRed and it worked very nicely. However as soon as we tried to add in some surface markers it became a compensation nightmare due to the changing signals and differing leakage into many other channels. I took advice from a fluorochrome guru I know, who suggested we use FuraRed alone and measure the calcium bound (increasing signal) off the violet laser and the calcium free (decreasing signal) off the blue. This would have the advantage of being more an accurate measurement of the flux since it is the loading of only a single dye and hopefully free up some parameters for the desired surface markers.
After the loading the cells with FuraRed alone, we stimulated with ionomycin and measured the signals in all 18 detectors to see which channels were affected. Curiously we found the decreasing signal to be greater off the green laser which I suspect is because the output of our 488nm is at 25mW whereas the 532nm is 150mW. Regardless, there were 4 parameters which were unaffected by the calcium flux: FITC, APC, AF700 and APC-Cy7, which would mean they could stain for the markers of interest and include a viability dye. Happy days.
They tried the full panel today (minus the live/dead) and got a very nice flux with ionomycin which did not affect the measurement of the surface markers - woo hoo! However, when they used their specific chemokine stimulant (which had previously shown a nice flux with Fluo-3), there was no detectable change in signal - boo! Which possibly explains why I couldn't find any papers using FuraRed alone......
So, does anyone have a good method for combining calcium flux detection with staining for surface markers?
FYI, we are using a SORP LSRII with the following configuration:
405nm (25mW) - PB, AmCyan, QD545, QD565, QD585, QD605, QD655 & QD705
488nm (25nm) - FITC, PerCP-Cy5.5
532 (150mW) - PE, PE-TxR, PE-Cy5, PE-Cy5.5 & PE-Cy7
633nm (50mW) - APC, AF700 & APC-Cy7
As ever, any advice would be gratefully received.
Helen Ferry, D.Phil
Flow Sorting Facility Manager
Experimental Medicine Division NDM
Level 5, Room 5605
John Radcliffe Hospital
+44 1865 228964
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