[Cytometry] Calcium flux + surface markers

Clintrial clintrial at gmail.com
Fri May 20 02:20:48 EDT 2011


What T cell markers did your user labeled? Did they label the receptor of
that chemokine, or some receptors that might interfere with GPCR signalling
pathway, or the receptor like CD3, which causes Ca2+ loading already. If the
percentage of that T cell subset is large enough, I would suggest run a
non-labeled control. If there is an influx, it should be detected by FACS.
Ionomycin works as a ionophore, which induces calcium influx by inserting a
pore specific for Calcium cation, a mechanism different from GPCR, which
triggers Ca2+ influx by store operated calcium channel.

I used to use multiple markers to label mouse lung DCs and measured the Ca++
influx induced by chemokine. overall the signal was not very good. My
speculation is that the labeling messed it up. However, in human
monocyte-derived DCs, I could record very good signal with Indo-1. I hope
this information helps.

Zhifei

On Thu, May 19, 2011 at 11:01 AM, Elizabeth R. Simons <esimons at bu.edu>wrote:

> 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, 1995
>
> 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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