We processed 20 μg of mitochondrial fractions using the Citrate Synthase Activity Kit (Sigma, St. Louis, MO). For subsequent assays of complex I–IV activity, we determined the citrate synthase activity in a sample of Tfam heterozygous mitochondria and adjusted amounts of wild-type and knockout littermate mitochondria to match that level. Complex I–IV were analyzed as detailed previously[25 (link)], using assay rates in the presence or absence of an inhibitor to determine the complex-specific activity.
Citrate synthase activity kit
Manufactured by Merck Group
Sourced in United States
The Citrate Synthase Activity Kit is a laboratory tool designed to measure the activity of the citrate synthase enzyme. Citrate synthase is a key enzyme in the citric acid cycle, a central metabolic pathway. The kit provides the necessary reagents and protocols to quantify citrate synthase activity in various biological samples.
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3 protocols using citrate synthase activity kit
1
Mitochondrial Complex I-IV Activity Analysis
2
Citrate Synthase Activity Assay
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Equal amounts of proteins (10 μg) from total extracts obtained by renal tissues or human RPTECs were analyzed with Citrate Synthase Activity Kit (Sigma-Aldrich) according to the manufacturer’s protocol. The citrate synthase activity was determined by the multimode microplate reader TECAN Infinite M200 PRO (Tecan Group Ltd.) at 412 nm under controlled temperature on a kinetic program for 1.5 min every 10 s.
3
Mitochondrial Respiration in RV Pressure Load
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Mitochondria were isolated from fresh RV tissue subjected to 12 weeks of pressure load by PAB, as described in the Supplemental methods. Mitochondrial respiration was measured by the oxygen consumption rate with either pyruvate and malate, or palmitoyl CoA and malate as substrate, in the presence of ADP in a stirred, 2-channel high-resolution respirometry (Oroboros, Innsbruck, Austra). The different states, including the ADP-driven state 3 (State 3) as well uncoupled state 3 (State 3u), representing mitochondrial conditions (ADP or respectively ATP-rich environment, and intact respectively absence of membrane gradient), were analyzed as described in the Supplemental methods. Oxygen consumption rate was corrected for protein content. Citrate synthase activity kit (Sigma Aldrich, USA) was used as a marker of mitochondrial density.
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