HI Umar,

I think that will depend on the cell type, the temperature and duration of incubation. Neurons almost certainly would not be very happy to be incubated in such high calcium. However, epithelial cells and fibroblasts are not excitable cells and they may be able to cope with it well.

best wishes

Refik

Dear Umar,

I suggest that you read the following paper:

Science 9 November 1979:

Vol. 206 no. 4419 pp. 700-702

DOI: 10.1126/science.386513

REPORTS

Calcium dependence of toxic cell death: a final common pathway

FA Schanne, AB Kane, EE Young, JL Farber

ABSTRACT

Primary cultures of adult rat hepatocytes were treated in the presence or absence of extracellular calcium with ten different membrane-active toxins. In all cases more than half the cells were killed in 1 to 6 hours in the presence but not in the absence of extracellular calcium. An effect of calcium on the primary mechanism of membrane injury by any of the agents cannot be implicated. Viability, as determined by trypan blue exclusion correlated well with other indices of viability such as plating efficiency and the hydrolysis of fluorescein diacetate. It is concluded that the cells are killed by processes that involve at least two steps. In each type of injury, disruption of the integrity of the plasma membrane by widely differing mechanisms is followed by a common functional consequence involving extracellular calcium, and most likely representing an influx of calcium across the damaged plasma membrane and down a steep concentration gradient. This later step represents, or at least initiates, a final common pathway for the toxic death of these cells.

Hoping this will be helpful,

Rafik

Hi Umar,

Intriguing traces. We find that most non-excitable cell types have stable cytosolic calcium levels when we change extracellular calcium providing that calcium influx pathways have not been activated. I guess an obvious things to consider is whether the cells have been sitting in calcium-free medium for a while, and the intracellular calcium stores have become depleted. That would engage store-operated calcium entry. That could account for the elevation of cytosolic calcium you see with 2 mM calcium medium too. Alternatively, the elevation of cytosolic calcium you see when adding the 2 and 20 mM solutions could occur via activation of the calcium-sensing receptor, which would also activate store-operated calcium entry.

Also, is your 20 mM calcium medium isotonic with the other medium you are applying? 20 mM is not that much of a change, but the cells could be reacting to an osmolarity difference.

Is the 20 mM calcium causing precipitation of calcium-phosphate particles? If so, they can be damaging to cells (Fetuin-A and albumin alter cytotoxic effects of calcium phosphate nanoparticles on human vascular smooth muscle cells. Dautova Y, Kozlova D, Skepper JN, Epple M, Bootman MD, Proudfoot D. PLoS One. 2014 May 21;9(5):e97565.)

Have you tried adding propidium iodide, or another live/dead cell stain, at the same time as the 20 mM calcium medium to see if there is any membrane damage? The fact that you can keep fluo4 inside the cells (suggested by the steady state fluorescence in your traces) would argue against membrane damage, but it's worth formally checking. 

Best of success,

Martin

I observed your graph and I think like Bootman tha in 400s in 0Ca probably the internal stores are partial or totaly depleted and replacing external Ca you are activating store-operated calcium entry (SOCE), that you see also with 2 mM Ca. Also be careful with the osmolarity of both solutions. To be sure this is the case you can try to block that Ca increase with 2-APB that blocks the store operated Ca entry. See also the paper on the effects of osmolarity changes on SOCE, J Muscle Res Cell Motil. 2013 Dec;34(5-6):379-93..

Dear Umar,

I agree with above comments. Pura is right that if you block your SOCE with 2-APB than you may see a significance between 0 Ca and 2 Ca already. This way may alow you to use lower concentrations of extracellular Ca 5 or 8, rather than 20 mM. Yes you need to keep an eye also on your osmolarity. If you are not adjusting your osmolarity that changes the membrane's structure and ionic permebility, you may add error to your results.

Best wishes,

Refik