Mitochondrial Citrate Tracing Assay

Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer^{3 (link)}. For citrate tracing, 40 µM [U-¹³C]-citrate, 40 µM NADH, and 40 µM CoA with or without 40 µM ATP and 50 µM ACLY inhibitor were added to the assay buffer. Mitochondria were incubated in the tracing buffer for 10 min, at 37 °C with 200 r.p.m. agitation in a heat block.

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Lee W.D., Mukha D., Aizenshtein E, & Shlomi T. (2019). Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells. Nature Communications, 10, 1351.

Publication 2019

Mitochondria Isolation Kit

Assay Buffer Citrate Egta Isolation M 200 Mitochondria Mitochondrial Mops Nadh Nutrients Pellets Tris

Corresponding Organization :

Other organizations : Technion – Israel Institute of Technology

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Variable analysis

independent variables

Presence or absence of ATP (40 µM)
Presence or absence of ACLY inhibitor (50 µM)

dependent variables

Mitochondrial metabolic activity

control variables

Mitochondria preparation method (magnetic beads method from Mitochondria Isolation Kit; Miltenyi Biotec)
Assay buffer composition (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4)
Nutrients and tracer added to the assay buffer (40 µM [U-13C]-citrate, 40 µM NADH, 40 µM CoA)
Incubation conditions (10 min, 37 °C, 200 r.p.m. agitation)

controls

Positive control: Mitochondria incubated with all nutrients and cofactors (40 µM [U-13C]-citrate, 40 µM NADH, 40 µM CoA, 40 µM ATP)
Negative control: Mitochondria incubated with all nutrients and cofactors except ATP (40 µM [U-13C]-citrate, 40 µM NADH, 40 µM CoA)

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