Complete tablets edta free

Mitochondrial Ca²⁺ uptake and ΔΨ_m were determined by simultaneous monitoring of cytosolic Ca²⁺ with Fura-2FF (0.5 μM; Life Technologies) and ΔΨ_m with the lipophilic cationic dye 5,5′,6,6′-tetrachloro-3,3′-tetraethylbenzimidazolcarbocyanine (JC-1; 800 nM; Life Technologies). After trypsinization and neutralization, cells were pelleted at 200 × g for 5 min, resuspended in 20 mL of phosphate-buffered saline, and pelleted at 150 × g for 2 min. 8 × 10⁶ cells were resuspended in 1.5 mL ICM buffer containing 40 μg/ml digitonin to permeabilize the cells, protease inhibitors (EDTA-free complete tablets; Roche Applied Science, Indianapolis, IN), 2 μM thapsigargin to block the SERCA pump, and 2 mM succinate to energize mitochondria (67 (link)). Fura-2FF (1 μM) was used as a cytosolic Ca²⁺ indicator. After 20 s of data recording, JC-1 was added. At 1200 s or 400 s CCCP, 10 μM, was added. Fluorescence was measured using a dual-wavelength spectrofluorometer (PTI) with 490-nm excitation and 535-nm emission for monomeric JC-1 and 570/595 nm for the J-aggregate. ΔΨ_m was calculated as the ratio of J-aggregate and the monomer (68 (link)).

Freshly isolated LV myocytes from WT and Tg26 mice were subjected to 2h of either normoxia or H/R. After gentle centrifugation, myocytes were transferred to an intracellular-like medium containing (in mM): 120 KCl, 10 NaCl, 1 KH₂PO₄, 20 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-Tris, 10 succinate, thapsigargin (2 ¼g/ml), digitonin (80 ¼g/ml), pH 7.2, and protease inhibitors (EDTA-free complete tablets, Roche Applied Science). Fura-FF (0.5 ¼M) was added at 0 s, and JC-1 (800 nM)(both from Molecular Probes) was added at 20s to measure extra-mitochondrial Ca²⁺ and ΔΨ_m, respectively. Fluorescence signals were monitored in a temperature-controlled (37°C) multi-wavelength excitation (ex) and dual wavelength emission (em) spectrofluorometer (Delta RAM, Photon Technology Int.) using 490-nm ex/535-nm em for the monomer, 570-nm ex/595-nm em for the J-aggregate of JC-1, and 340- and 380-nm ex/510-nm em for Fura-FF. The ratiometric dye Fura-FF was calibrated as previously described (Mallilankaraman et al. 2012 (link), Miller et al. 2014 (link)). At 450 s, 10 ¼M Ca²⁺ pulse was added, and ΔΨ_m and extra-mitochondrial Ca²⁺ were monitored simultaneously. ΔΨ_m was calculated as the ratio of the fluorescence of the JC-1 oligomeric to monomeric forms. Cytosolic Ca²⁺ clearance rate was taken to represent mitochondrial Ca²⁺ uptake.