I am stimulating my cultured cells and recording calcium activity as fluorescence change using live-cell calcium imaging. Though I am more concerned about the decay of the signals, for which I am calculating Tau ( the time constant of decay).
Additionally, I am interested to examine the Rise of Signal. What would be the best parameter to study a rising signal? For example, a few measures of a rising signal are Rise time, Time constant, Slope ..................etc...
P.S. Not having a mathematical background :P
Hi Tushar,
Unless your experimental settings allow you to differentiate between the influx of calcium from the plasma membrane and the release of calcium from internal stores, your overall calcium signal will be a mix of both further affected by calcium extrusion and repumping. Therefore, a good start would be to look at the "area under the curve" which gives you an estimate of the overall calcium load.
You can of cause further dissect the various components of your calcium signal by measuring rise time, peak amplitude, decay time, half width, etc... again keeping in mind that all these parameters are affected by a mix of several phenomena occurring concomitantly.
Dear Tushar: The rising Ca signal would depend on Ca influx from the exterior, Ca release from internal store, or both. One might use tau (time constant) to measure the rising rate of Ca signals.
Hi Tushar,
Unless your experimental settings allow you to differentiate between the influx of calcium from the plasma membrane and the release of calcium from internal stores, your overall calcium signal will be a mix of both further affected by calcium extrusion and repumping. Therefore, a good start would be to look at the "area under the curve" which gives you an estimate of the overall calcium load.
You can of cause further dissect the various components of your calcium signal by measuring rise time, peak amplitude, decay time, half width, etc... again keeping in mind that all these parameters are affected by a mix of several phenomena occurring concomitantly.
Would it be possible to measure the "normalized" area under the curve for the fluorescence signal? For the normalization you can use the fluorescence response (area) in non-stimulated cells (where you only have the influx) for the measurement.
Hello,
What do you do to stimulate your cells in culture? are you patching individual cells? What sort of cells do you have in culture?
Hi Tushar,
I completely agree with above observations by Norbert Weiss. The measured cell Ca results from a steady-state of various processes that move of Ca in and out of the cytosol. I would suggest trying to sort them out to characterize each one. For instance, you may start by adding EGTA to block Ca influx, or thapsigargin to inhibit Ca release from endogenous stores (mainly endoplasmic reticulum), and a plasma membrane Ca pump blocker (vanadate may be worth trying). I think your results would be more meaningful in that way. Good luck. Pedro.
Norbert Weiss Thank you so much for the reply,
I agree that its good start by looking at the area under the curve.
Since my experimental settings don't allow me to differentiate between release of calcium from various sources, I am broadly quantifying the rise (using e-folding time or time constant, Tau, for the rise) (as suggested by Sheng-Nan Wu and decay (Time constant, tau) by fitting it to a single exponential.
I appreciate the responses from peers !!
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