Fig. 3. Astrocytic SOCE is mediated by Orai channels in combination with Stim1. (a) Schematic illustration of astrocyte Ca2+ imaging in inverted microscope using ratiometric Ca2+ dye, Fura-2 AM. (b) Experimental protocol of Ca2+ imaging. Ca2+ free HEPES solution containing 200 nM EGTA was bath applied for 1200 s. Meanwhile, 1 µM thapsigargin, the SERCA inhibitor, was applied for 120 s after 60 s baseline to induce Ca2+ release from ER. External solution was changed to the normal HEPES solution containing 2 mM Ca2+ at 1200 s. (c) Left top, Fura-2 AM loaded cells. Left bottom, Orai1, Orai2, and Orai3 shRNA co-transfected cells expressing mCherry or EGFP. Right top, representative Ca2+ imaging 340/380 ratio images taken at 1 min, 3 min, 20 min, 25 min. Right bottom, representative 340/380 ratio traces of two cells. Orai1/2/3 shRNA transfected cell is indicated with red mark and endogenous control was indicated with yellow mark. (d~j) Average of total Ca2+ imaging traces and scatter plots of ER Ca2+ release (Release), store operated Ca2+ entry (Entry) and Entry/Release ratio (Ratio) of primary cultured astrocytes transfected with scrambled shRNA (d), Orai1 shRNA (e), Orai2 shRNA (f), Orai3 shRNA (g), TrpC1 shRNA (h), Orai1/2/3 shRNA (i), Stim1 shRNA (j). Scale bar indicates 0.5 for y axis and 100s for x axis. Tg: thapsigargin.
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