Tcs sp5 confocal laser system

Cytosolic Ca²⁺ ([Ca²⁺]_i) was measured as previously described (Shen et al. 2011). In brief, cells were incubated with 10 μmol/L Fluo‐8/AM and 0.02% pluronic F‐127 (Invitrogen, Carlsbad, CA) for 40 min in the dark at 37°C. Ca²⁺ stores were depleted by treating cells with 4 μmol/L TG for 10 min in 0Ca²⁺‐PSS. Ca²⁺ influx was initiated by applying 1 mmol/L extracellular Ca²⁺. Cells were pretreated with scrambled siRNA or Orai1 siRNA for 24 h before experiments. Fluorescence signal was recorded by Leica TCS SP5 confocal laser system. Changes in [Ca²⁺]_i were displayed as the ratio of fluorescence relative to the intensity before applying extracellular Ca²⁺ (F1/F0).

Immunofluorescence was performed as described elsewhere [31 (link)]. Cultured aortae or primary cultured VSMCs were fixed with 4% formaldehyde for overnight or 10 min respectively, followed by permeabilization with 0.1% Triton X-100 dissolved in PBS. The samples were blocked by 2% BSA at room temperature for 1 h before incubating with primary antibody at 4 °C overnight. After washing with PBS for three times, the samples were incubated with donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) for 1 h at room temperature and mounted in 90% glycerol in PBS and the fluorescent signals were determined by a TCS SP5 confocal laser system (Leica, Germany). The 8-bit images were analyzed with ImageJ software [32 (link)]. Briefly, projected images were generated by collecting maximum pixel intensity of the in-focus frames into a single frame. The images were threshold to remove background fluorescence and used to create a mask image. Using the mask images, number of STIM1 puncta was scored with automatic “Analyze Particles” algorithm of ImageJ software and using cluster size of 3–100 pixels and circularity 0.1–1.0.

[Ca²⁺]_i measurements were performed as previously described4 (link)9 (link). Briefly, MAECs seeded on rectangular coverslips were loaded with 10 μM Fluo-8/AM and 0.02% pluronic F-127 for 30 min. The coverslip with the cells was mounted in a parallel plate flow chamber. The flow was initiated by pumping normal physiological saline solution (NPSS) containing 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl_2, 10 mM glucose, 5 mM HEPES, and 1% BSA (pH 7.4). The fluorescence signals were recorded and analyzed using a Leica TCS SP5 confocal laser system, and [Ca²⁺]_i changes were displayed as a ratio of fluorescence (F₁/F₀). The endothelial cells were challenged with 4α-PDD (5 μM) or the shear stress induced by flow that was approximately 5 dyne/cm².

HEK-293T cells transfected with pFJ-EA, pFJ-K15P, and pFJ-K15P(Y⁴⁸¹F) or EA.hy926 cells infected with lentiviral vector, lentivirus K15P(lenti-K15P), and lentivirus K15P (Y⁴⁸¹F)[lenti-K15P (YF)] were seeded on circular glass coverslips in 1.5 mL DMEM for 24 h and the cells were 70–80% confluent before measurement. At the time of the experiment, the cells were loaded with 10 μmol/L Fluo-8/AM at 37 °C for 30 min. Ca²⁺ stores were depleted by treating cells with 4 μmol/L thapsigargin (TG) for 10 min in Ca²⁺-free phosphate-buffered saline (PBS), which contained 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl₂, 10 mmol/L glucose, 0.2 mmol/L EGTA, and 5 mmol/L HEPES, pH 7.4. Ca²⁺ influx was initiated by applying 2 mmol/L extracellular Ca²⁺. Fluorescence was recorded using a Leica TCS SP5 confocal laser system (Heidelberg, Germany). Changes in cytosolic Ca²⁺ [Ca²⁺]_I were displayed as the ratio of fluorescence relative to the intensity before the application of extracellular Ca²⁺ (F1/F0).