I am trying to study calcium ion influx in response to hormone stimulation in adherent human cell line using calcium green AM-1, a calcium ion binding dye by flowcytometry. Can anybody help me out with the protocol for the same?
Since this is adherent cell line is it ok to trypsinized the to take out from the flask?
How to determine the optimum calcium binding dye concentration??
What kind of graphical representation one can expect??
(Is it like influx will shoot up slowly and slowly and and will drop down within few minutes or it will remain maintain throughout??)
Maybe this protocol could help..
http://www.bio-protocol.org/e910
Thank you Cassio
Howere still some questions remained unanswered as in the bio-protocol the cell cell line used was Jurkat (non-adherent cells/ suspension culture).
1. Does trypsin-EDTA used to detached adherent cells from the flask will affect the calcium ion influx which could be mediated through a stimulatent known to act through surface receptor???
2. Is it necessary to stimulate cells with thapsigargin initially, one of the calcium ion inhibitor for esdoplasmic reticulum prior inducing cells with stimulent which could be hormone???
Thank you.
Dear Sush Sab,
I think that a short Trypsin -EDTA treatment will not affect the potential of a surface receptor to induce its signalling pathway, in the context of trypsin cleaving the receptor and thus impairing the pathway.
In our lab we have studied calcium influx in macrophages and the required detachment prior to measurement did not impact the calcium influx triggered through the TLR4-CD14 mediated patway.
As far as the dynamics of the calcium signal are concerned i think it depends on the pathway you study. Treating cells wit TG may result in an increased signal as calcium ions will not be able to get pumped into the ER, but it may not give you information about the kinetics of the signal under control/untreated conditions.
Best regards,
Nikos
Dear Nikos
Thank you so much.
I am working with human cell line (adherent) and interested in measuring calcium ion influx by flowcytometry using Calcium Green AM-1 calcium binding dye.
I could see the significanct increase in calcium ion influx in response to IONOMYCIN (log scale).
However, when the cells were stimulated with homone variable concentrations i didn;t find significant difference though not challenged with TG which is very surprising since cells are hormone responsive.
I request you kindly send me a detail protocol specially buffer used and its composition. And also wish to know it is necessary to maintain the temparature of the sample tube at 37 degree centigrade while aquisition (Specially when Room Temp is 19-22 degree centigrade where flow instrument is installed).
Thanking you.
Dear Sush Sab,
I am attaching a pubmed link, were you can find the experimental procedure we have followed in detail. Generally, depending on the signal to be measured and the stimulant used a representation in the log or linearscale should be tested.
Ionomycin response, serving as a positive control can be beautifully plotted in a log scale whereas i found that when dealing with other physiological pathways the linear scale can provide more information.
This may be the reason that you can not detect any measurable response. In addition the availability and expression of the target receptor in the cells used may influence the intensity of the produced signal i.e it may be big enough to induce the downstream pahtway but not adequate to be measured with flow cytometry.
Last but not least, i do not think that maintaining the temperature at 37 is mandatory, as the dynamics of the signal are pretty fast and you should see the increase and recession of the signal during a 5 min period.
http://www.ncbi.nlm.nih.gov/pubmed/?term=Papaioannou+N+E+et+al+2016
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