Ix83 research inverted microscope

pH and calcium clamping were carried out using CalipHluor^mLy. Fibroblast cells were pulsed for 1 h and chased for 2 h with 500nM CalipHluor^mLy. Cells are then fixed with 200 mL 4% paraformaldehyde (PFA) for 15 min at room temperature, washed three times and retained in 1× PBS. To obtain the intracellular pH and calcium calibration profile, endosomal calcium concentrations were equalized by incubating the previously fixed cells in the appropriate calcium clamping buffer [HEPES (10 mM), MES (10 mM), sodium acetate (10 mM), EGTA (10 mM), KCl (140 mM), NaCl (5 mM) and MgCl₂ (1 mM)] by varying amount of free [Ca²⁺] from 1 μM to 10 mM and adjusted to different pH values. The buffer also contained nigericin (50 μM), monensin (50 μM) and ionomycin (20 μM) and the cells were incubated for 2 h at room temperature.
For realtime pH and calcium measurements, fibroblast cells are pulsed with 500nM of CalipHluor^mLy for 1 h, chased for 9 h (8 h for L0625 cells) and then washed with 1× PBS and imaged in Hank’s Balanced Salt Solution (HBSS). Imaging was carried out on IX83 research inverted microscope (Olympus Corporation of the Americas, Center Valley, PA, USA) using a 100X, 1.42 NA, DIC oil immersion objective (PLAPON, Olympus Corporation of the Americas, Center Valley, PA, USA) and Evolve Delta 512 EMCCD camera (Photometrics, USA).

Micropillar stamps with concave tip curvature were incubated in 0.2% rhodamine B in 1X PBS for 16 h. These stamps were then dried and rinsed with 70% ethanol in DI water. The pillars were then imaged via confocal microscopy on an Olympus IX83 Research Inverted Microscope (10X objective, N.A = 0.4; 40X objective, N.A. = 0.95) to generate Z-stack images.