[Cytometry] Calcium flux + surface markers

Elizabeth R. Simons esimons at bu.edu
Thu May 19 14:01:07 EDT 2011

If you have a uv 357nm laser, try using indo-1 instead of the fura  
combination - it's a lot more sensitive, a single probe (less is  
always better because the more cytoplasmic probes you add as esters  
the more you acidify the cytoplasm), and has no overlap with your  
other probes. Protocols using it and various surface markers  
simultaneously are in several publications from my lab (try  
Brunkhorst et al and Strohmeier et al, refs below).
Elizabeth R. Simons

B. Brunkhorst, K.G. Lazzari, G.Strohmeier, G.Weil and E.R. Simons.  
Calcium Changes in Immune Complex-stimulated Human Neutrophils:  
Simultaneous Measurement of Receptor Occupancy and              
Activation Reveals Full Population Stimulus Binding but Subpopulation  
Activation, J.Biol.Chem. 266:13035-13043, 1991.
B.A. Brunkhorst, G. Strohmeier, K. Lazzari, G. Weil, D. Melnick, H.B.  
Fleit and E.R. Simons, Differential roles of FcgRII and FcgRIII in  
Immune Complex Stimulation of Human Neutrophils,  J.Biol.Chem.  
267:20659-20666, 1992
G.R. Strohmeier, B.A. Brunkhorst, K.F. Seetoo, G.J. Weil and E.R.  
Simons. Neutrophil Functional Responses Depend Upon Immune Complex  
Valency. J. Leuk.Biol. 58:403-414, 1995
G.R. Strohmeier, B.A. Brunkhorst, T. Meshulam, J. Bernardo, K.F.  
Seetoo and E.R. Simons, The Role of FcgR subclasses. FcgRII and  
FcgRIII, in the Activation of Human Neurophils by Low and  
High             Valency Immune Complexes. J. Leuk. Biol. 58:415-422,  
On May 19, 2011, at 12:16 PM, Helen Ferry wrote:

> Dear Flowers
> 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.
> Best wishes
> Helen
> Helen Ferry, D.Phil
> Flow Sorting Facility Manager
> Experimental Medicine Division NDM
> Level 5, Room 5605
> John Radcliffe Hospital
> Headley Way
> Headington
> Oxford
> OX3 9DU
> United Kingdom
> +44 1865 228964
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Elizabeth R. Simons, Ph.D.
Professor of Biochemistry
Boston University School of Medicine
80 E. Concord Street
Boston, MA 02118
(617) 638-4332 phone
(617) 638-5339 FAX
esimons at bu.edu
ersimons at earthlink.net

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