Two mg of isolated mitochondria were suspended in a total volume of 1 ml consisting KCl buffer, 1 mM Malic acid (Sigma-Aldrich), 7 mM Pyruvate (Sigma-Aldrich), and 50 nM Calcium Green 5N (Invitrogen) in a quartz cuvette, which was placed inside the fluorimeter (PTI QuantaMaster 800, Horiba Scientific). Calcium uptake was measured by fluorescent emission of Calcium Green 5N. Simultaneously, mitochondrial swelling was measured by transmittance light. Each BH3 mimetic was used at concentrations: 0.5, 1, 10, 50, 100 nM, and 1 µM. Final BH3 mimetics concentrations used throughout the study were 200 nM ABT-199 (Selleckchem.com , S8048), 100 nM A-1331852 (abcam, ab218112), 100 nM ABT-737 (EMD Millipore Sigma, 197333), 10 nM S63845 (Selleckchem.com , S8383), 10 nM Obatoclax (Selleckchem, GX15-070). For some experiments ADP (300 uM) (Sigma-Aldrich, A2754) and/or CsA (2 uM) (Sigma-Aldrich, 30024) were used to desensitize the MPTP. CaCl2 (20 μM, 40 μM, and 80 µM) (Sigma-Adrich, C4901) was added into this system in succession until MPTP opening occurred, indicated by an upturn in Calcium Green 5N fluorescence, or when mitochondria were saturated with Ca2+ and were no longer able to take up further additions of CaCl2, indicated by a stair-stacking in the Ca2+ uptake graphs.
Pti quantamaster 800
Manufactured by Horiba
Sourced in Germany
The PTI QuantaMaster 800 is a fluorescence spectrometer designed for advanced photoluminescence measurements. It features a high-performance dual-monochromator system and a versatile optical layout to support a wide range of sample types and experimental configurations.
Automatically generated - may contain errors
Lab products found in correlation
Malic acid, by Merck Group (1 mentions)
Calcium green 5n, by Thermo Fisher Scientific (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Abt 199, by Selleck Chemicals (1 mentions)
Obatoclax, by Selleck Chemicals (1 mentions)
A 1331852, by Abcam (1 mentions)
A2754, by Merck Group (1 mentions)
Calcium green 5n, by Merck Group (1 mentions)
2 protocols using pti quantamaster 800
1
Mitochondrial Calcium Uptake and Swelling Assay
2
Assaying NBD-PS Lipid Flipping
Check if the same lab product or an alternative is used in the 5 most similar protocols
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To assay the flipping of NBD‐PS, proteoliposomes or protein‐free liposomes (10 μL) were incubated for 30 min at 30°C with 15 μL of reconstitution buffer supplemented with 2 mM ATP and 3 mM MgCl2 or NaCl, to reach a final concentration of 1.2 mM ATP and 1.8 mM MgCl2 or NaCl. Subsequently, the samples were diluted to 1 mL of reconstitution buffer, transferred into cuvettes (10 mm light path cuvette, macro, 4 clear sided; Brand, Wertheim, Germany), and measured at room temperature in a fluorometer (PTI‐Quantamaster 800, Horiba, Benzheim, Germany). Fluorescence traces were recorded at λex/λem 467/537 nm, slit width 5 nm, resolution 0.1 s. Sodium dithionite (laboratory reagent grade, Thermo Fisher Scientific, 0.5 M in 0.9 M non‐buffered Tris) was added after 50–60 s to 10 mM final concentration. For complete reduction of all NBD‐PS analogs by dithionite, Triton X‐100 was added to a final concentration of 0.5% (w/v). Data analysis was performed as described above.
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