Cytochrome c release from mitochondria to cytoplasm was evaluated using a fluorescence microscope (DMI 4000 B Leica Microsystems, Bannockburn, IL) coupled to the software LAS-AF. The control and 4-NRC-treated cells (1 × 106 cells/mL) were fixed with methanol and then seeded on BD cell-takTM-coated coverslips. Cells were permeabilized and blocked with PBS solution containing BSA (1%, p/v) and Tween 20 (0.1%, v/v) in humidified chamber for 1 h, at room temperature. Then the cells were incubated with cytochrome c antibody for 1 h and nuclei were stained with Hoechst 33258. The cells were then washed three times with PBS and mounted, using an appropriate mounting medium (PBS with 90% glycerol, v/v).
Cell taktm
Manufactured by BD
The BD cell-takTM is a manual cell harvesting device designed for use in cell culture laboratories. It enables the efficient detachment and collection of adherent cells from cell culture vessels, facilitating further downstream processing and analysis.
Automatically generated - may contain errors
2 protocols using cell taktm
1
Cytochrome c Translocation Microscopy
2
Pioglitazone Effects on Mitochondrial Function
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MES GSCs plated at 105 cells/well in 6-well plates were treated with 10 μM pioglitazone for 48 h. Cells were then transferred to a Seahorse microplate (Agilent) precoated with BD Cell-TakTM (BD Biosciences, San Jose, CA) following the manufacturer’s instructions. Mitochondrial stress tests were performed as recommended by the manufacturer (Agilent). The basal oxygen consumption rate was measured, and then it was measured again after the following drug injections: 2 μM oligomycin, 0.5 μM FCCP, and 0.5 μM rotenone/antimycin A. The reads were normalized by total protein amounts measured with a Pierce BCA Protein Assay kit (Thermo).
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