Measuring Intracellular Calcium in hTM/pTM Cells

Cultured hTM/pTM cells were loaded with 5 μM Fura-2-AM for 15–30 minutes and were perfused with isotonic saline (pH 7.4) containing (in mM): 98.5 NaCl, 5 KCl, 3 MgCl₂, 2 CaCl₂, 10 HEPES, 10 D-glucose, 93 mannitol. Epifluorescence imaging was performed as described22 (link)23 (link) using inverted Nikon Ti or upright Nikon E600 FN microscopes with 20x (0.75 N.A. oil) and 40x (1.3 N.A. oil & 0.8 N.A. water) objectives and Nikon Elements software. In a subset of experiments, [Ca²⁺]_pTM levels were calibrated using the standard equation (K_d at RT = 224 nM). Results represent averages across cells (3–6 slides, each containing 20–40 cells) from at least three separate experiments.