Primary rat neurons or human i3Neurons were plated on Seahorse XFp plates (Agilent) at a density of 40,000 cells/well, then either treated with 2DG or transduced with codon-optimized lentiviruses as described in previous sections. 48 h after 2DG treatment or 4 days after transduction, the Seahorse Extracellular Flux assay was performed using the Seahorse XF Mini instrument (Agilent) according to the manufacturer’s instructions. During the assay, the cells were treated sequentially with each of the following mitochondrial toxins: oligomycin (1.5 μg/ml), FCCP (3 μM), and antimycin (1 μM) (all Cayman Chemical). Immediately after the assay, the cells were lysed with RIPA buffer with protease inhibitor (Thermo Scientific). The lysates were analyzed for total protein content using the Pierce BCA protein assay (Thermo Scientific). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) values were then normalized to total protein.
Seahorse xf mini instrument
Manufactured by Agilent Technologies
The Seahorse XF Mini instrument is a compact and versatile tool designed for metabolic analysis. It measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells, providing insights into their energy metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Oligomycin, by Cayman Chemical (2 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Cayman Chemical (2 mentions)
Antimycin, by Cayman Chemical (2 mentions)
Seahorse xfp plates, by Agilent Technologies (2 mentions)
Ripa buffer with protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (2 mentions)
2 protocols using seahorse xf mini instrument
1
Mitochondrial Function Profiling in Neurons
2
Mitochondrial Function Assay in Neurons
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Primary rat neurons or human i3Neurons were plated on Seahorse XFp plates (Agilent) at a density of 40,000 cells/well, then either treated with 2DG or transduced with codon-optimized lentiviruses as described in previous sections. 48 hours after 2DG treatment or 4 days after transduction, the Seahorse Extracellular Flux assay was performed using the Seahorse XF Mini instrument (Agilent) according to the manufacturer’s instructions. During the assay, the cells were treated sequentially with each of the following mitochondrial toxins: oligomycin (1.5 μg/ml), FCCP (3 μM), and antimycin (1 μM) (all Cayman Chemical). Immediately after the assay, the cells were lysed with RIPA buffer with protease inhibitor (Thermo Scientific). The lysates were analyzed for total protein content using the Pierce BCA protein assay (Thermo Scientific). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) values were then normalized to total protein.
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