I am trying to monitor calcium signals with ATP and TRPv4 in endothelial cells (bEnd.3). For this i incubate my cells with 3-5uM Fluo-4AM in 1X HBSS (pH 6.8).after 30mins, i wash with HBSS twice and then place them under microscope for observation. at this setting, i mostly find that cells are bright and clearly labelled with the dye(including the nucleus). if the cells look like this, after 5-10 mins baseline i add ATP 1-10uM and clearly see cellular response as a spike (well established).
the issue is the following: sometimes, only the cytosol is filled with the dye and is dimmer than the situation above. the nucleus looks clearly unmarked. As soon as i start imaging, i see that one region of my well becomes brighter and a wave of calcium flows through making them much brighter than before. These events are not predictable. All experiements are done at RT because i am working with channels that are temperature sensitive. i have tried to change Buffer to the live-cell imaging buffer and PBS (ph 7.4), tried to aspirate the medium without touching the bottom but this issue is still observable at times. what could be a possible explanation for this?? i wonder if the concentration of fluo4AM may play a role? again, these effects are not reproducible between different dishes on the same day and in the same experiment as such and happen randomly.
What you’re seeing is a mix of dye-loading variability and real biology. Fluo-4 AM loading depends on membrane permeability, intracellular esterase activity, temperature, pH/osmolarity, and efflux pumps. At higher loading (3–5 µM, 30 min, pH 6.8, RT), more AM ester enters and can de-esterify broadly, sometimes giving bright nuclear signal; at other times, lower esterase activity/greater efflux (MDR/organic anion transporters) yields dimmer, cytosol-restricted labeling with nuclear exclusion. Acidic HBSS (pH 6.8) and RT also slow de-esterification and alter Ca²⁺ handling, adding dish-to-dish variability. High intracellular dye can buffer Ca²⁺ (“calcium clamp”), changing responsiveness and baseline.
The unpredictable “wave” is classic for paracrine ATP-mediated Ca²⁺ waves and/or mechanosensitive activation: a small cluster of stressed cells releases ATP (via pannexin/connexin hemichannels), which propagates through P2Y receptors as an IP₃-dependent Ca²⁺ wave across the field. Triggers include slight mechanical disturbances (pipetting, stage movement, vibration), solution changes (osmolarity or pH jumps), local hypoxia, phototoxicity at the start of imaging, and activation of mechanosensitive channels such as TRPV4—especially at RT and with non-physiological pH. Once a “leader” region fires, the wave sweeps the well, making cells abruptly brighter.
Practical fixes and controls: use a physiologic buffer (HBSS or L-15) at pH 7.3–7.4 with Ca²⁺/Mg²⁺ and matched osmolarity; load lower dye (1–2 µM) with ≤0.02% Pluronic F-127 and ≤0.1% DMSO; after loading, allow a 15–20 min de-esterification in dye-free buffer; consider 1–2 mM probenecid to limit anion-transport efflux; if channel temperature-sensitivity permits, do the loading at 37 °C and then image at RT; minimize light intensity at start and avoid touching the dish or abrupt fluid flow. To confirm mechanism, abolish waves with apyrase (scavenges ATP) or P2 blockers (e.g., suramin/PPADS), reduce extracellular Ca²⁺/add EGTA, or block TRPV4 (e.g., HC-067047). If those suppress the phenomenon, your “random” events are bona fide mechanically/ATP-driven Ca²⁺ waves rather than an artifact of the dye.
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