The OCR (oxygen consumption rate) was measured using an XF HS Mini Analyzer (Seahorse Bioscience). Following the pulsing treatment, B16F10 cells were seeded into XFp cell culture 8-well mini plates in duplicate at a density of 3 × 103 cells/well. The cells were then cultured under standard conditions for 15 h. Before measurement, the medium was replaced with Seahorse XF Assay Media (Agilent, Santa Clara, CA, USA) with a pH of 7.4. The assay media was supplemented with 10-mM glucose, 2-mM L-glutamine, and 1-mM pyruvate. For the mitochondrial stress test, the following inhibitors were used at the indicated final concentrations: 1.5-μM oligomycin, 1-μM FCCP, and 0.5-μM rotenone–antimycin A. Two wells without cells were included to assess non-cellular oxygen consumption, and the value of non-cellular oxygen consumption was subtracted from the cellular OCR value. After completing the experiment, the OCR data were normalized to the number of cells.
Xf hs mini analyzer
Manufactured by Agilent Technologies
Sourced in United States, Germany
The XF HS mini analyzer is a compact, high-performance instrument designed for cellular metabolic analysis. It measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells, providing insights into their metabolic activity and function.
Automatically generated - may contain errors
Lab products found in correlation
Cell mito stress kit, by Agilent Technologies (2 mentions)
Cell tak, by Corning (2 mentions)
Mito test kit reagents, by Agilent Technologies (2 mentions)
Seahorse xf assay media, by Agilent Technologies (1 mentions)
Xfp cell culture miniplate, by Agilent Technologies (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Seahorse xf dmem media, by Agilent Technologies (1 mentions)
Seahorse xfp cell mito stress kit, by Agilent Technologies (1 mentions)
Seahorse xf cell mito stress test kit, by Agilent Technologies (1 mentions)
8 well cell plate, by Agilent Technologies (1 mentions)
Seahorse xf calibrant solution, by Agilent Technologies (1 mentions)
12 protocols using xf hs mini analyzer
1
Mitochondrial Respiration Profiling in B16F10 Cells
2
Mitochondrial Function Assessment in COCs
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The effects of 2 h FSH culture on mitochondrial respiration were also assessed with a Seahorse XF HS Mini Analyzer in the presence of mitochondrial drugs. COCs were cultured for 2 h with or without FSH in Seahorse XFp Cell Culture miniplates (20 COCs/well in triplicate, n = 5) in 175 µl of sterile XF assay buffer (as detailed above) at 37 °C. The miniplate was then centrifuged for 3 min at 300 g for cell adherence and put in the extracellular flux analyzer for OCR and ECAR measurements over 2.5 h. After four measurement cycles (as described above), mitochondrial drugs were loaded through the four injection ports of the cartridge at the specific times indicated in Fig. 4 A. A mitochondrial stress test was performed by sequentially injecting the following drugs: oligo (ATP synthase inhibitor, 1 µM), Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP, mitochondrial uncoupler, 1 µM), and rotenone + antimycin A (Rot + AA, complex I and III inhibitors, 0.5 µM each). The OCR and ECAR were measured in pmol/min and mpH/min, respectively, and were normalized by DNA concentration, as described above.
3
Measuring Bacterial Basal Respiration
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Bacterial basal respiratory activity was quantified using the Seahorse XF HS Mini Analyzer according to the manufacturer’s instructions. S. aureus was diluted 1:200 from overnight cultures in LB and grown to mid-exponential phase at 37°C. Cultures were diluted to OD600 = 0.025 and 180 μL of diluted cells was dispensed into each well of cell culture microplates precoated with poly-D-lysine (glycolysis stress test kit). The seahorse XF sensor cartridge was hydrated in a non-CO2 37°C incubator with sterile water (overnight) and pre-warmed XF calibrant for 60 min prior to measurement. Each bacterial strain was enumerated to confirm equal concentrations and run on the machine. Mixing and measurement time was set to three minutes in each cycle.
4
Seahorse XF HS Mini Analyzer Protocol
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Hepatocytes were seeded at a density of 40,000 cells per well in wells B–G on the miniplates, and wells A and H were left as blank controls. A side-by-side comparison was made between 7L vs 7F or 14L vs 7F7L in triplicate on the same miniplate. After the completion of their corresponding timelines, hepatocytes were cultured in XF Dulbecco’s modified Eagle medium (103575-100; Agilent Technology) supplied with 10 mmol/L glucose (103577-100; Agilent Technology), 1 mmol/L pyruvate (103578-100; Agilent Technology), and 2 mmol/L L-glutamine (103579-100; Agilent Technology) for 1 hour in a non-CO2 37°C incubator. Solutions (10×) were made for oligomycin (200 μmol/L), FCCP (80 μmol/L), and Rot/AA (50 μmol/L) as optimized final concentrations based on the earlier titration experiment and loaded to injection ports A–C on a hydrated cartridge. Subsequently, the XFp Cell Mito Stress Test protocol was executed on the Seahorse XF HS mini Analyzer. The protocol consisted of an equilibration period, basal OCR measurement (3 cycles, 6-minute interval), and 3 OCR measurements after drug injections each with 3 cycles and 6-minute intervals.
5
Respiratory Profile of Pancreatic Cell Lines
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Sphere-derived Panc185, PancA6L and Panc215 cells were plated in XF HS Miniplates (Seahorse Bioscience) at a cellular density of 5,000 cells/well. For OCR determination, cells were incubated in Seahorse XF DMEM media (103680, Agilent) supplemented with 2mM glutamine, 10mM glucose, and 1mM pyruvate for 1 h, prior to the measurements using the Seahorse XFp Cell Mito Stress Kit (103010, Agilent). After an OCR baseline measurement, the minimum oxygen consumption was determined adding 1.5µM oligomycin (O) and the maximal respiration rate was assessed by adding 1µM FCCP (F). At the end of the experiment the non-mitochondrial oxygen consumption was evaluated adding both 0.5µM rotenone (R) and antimycin (A). Experiments were run in a XF HS Mini analyzer (Seahorse Agilent), and raw data were normalized to total protein using BCA protein assay kit (Cat. no. 23225, Thermo Scientific).
6
Mitochondrial Respiration Assay
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Oxygen consumption rate was assayed using Cell Mito Stress kit (Agilent) and analyzed on Seahorse XF HS mini analyzer.
7
Mitochondrial Respiration Assay
8
Mitochondrial Respiration in γδ-T Cells
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OCR and ECAR were measured with a Seahorse XF HS Mini Analyzer using a Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies Inc., Santa Clara, CA, USA). Briefly, after thawing, γδ-T cells were cultured in RPMI 1640-10% FCS medium. The TCR stimulation time was 72 h. After washing with assay medium, γδ-T cells were plated at 2.0–3.0 × 105 cells/well, with the same number of cells included in each experiment, in a cell culture plate with assay medium and cultured for a total of 50–60 min at 37°C in a CO2-free incubator until analysis. The final concentrations of oligomycin, FCCP, and rotenone/antimycin A were 1.5, 1.0 μM and 0.5 μM, respectively. The basal OCR/basal ECAR ratio was calculated using the equation: (pmol/min)/(mpH/min).39 (link)
9
Measuring Metabolic Flux in Fly Brains
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OCR and ECAR were obtained using Agilent Seahorse XF HS Mini Analyzer as previously described [33 (link)]. Prior to the experiment, an Agilent Seahorse cartridge was hydrated overnight at 25°C with 200 μl of calibrant solution from Agilent. The following day, brains from adult flies were dissected in phosphate buffered solution (PBS) then immediately transferred into an 8-well cell plate from Agilent. Wells 1 and 6 were used as negative controls containing only 200 μl of Agilent Seahorse assay media supplemented with 10 mM glucose and 10 mM sodium pyruvate. Using dull forceps, each brain was positioned at the bottom of the well, centrally located between 3 raised spheres. Three brains per condition were used as it is the maximum number of samples that can be loaded within the same plate. The tissue restraints were gently lowered down using dull forceps. The tissue restraints were designed by Neville and colleagues [33 (link)] and manufactured by the Instrument Design and Fabrication Core Facility at Arizona State University. Basal OCR and ECAR measurements were collected from 6 cycles.
10
Mitochondrial Respiration Analysis of Breast Cancer Cells
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Cells were treated with PGV-1 for 24 h in a 35-mm culture dish. Following trypsinization, MDA–MB-231 (2.25 × 105 cells) and HCC1954 (1.5 × 105 cells) cells were dissolved in assay medium containing 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate and equally distributed in a Cell-Tak (Corning, USA, Cat # 354240) precoated miniplate. After spinning down the 8–well plate for cell adhesion, it was incubated for 30 min. Mito test kit reagents (Agilent, Germany, Cat #103010–100) containing FCCP (1 µM), oligomycin (1.5 µM), and rotenone/antimycin (0.5 µM) were injected into the cartridge, then the oxygen consumption rate (OCR) was recorded using an Agilent XF HS mini analyzer. Finally, the cells were measured for total protein to normalize the OCR data and later analyzed using the Seahorse Analytics platform (https://seahorseanalytics.agilent.com/# ) (Novitasari et al., 2023a (link), Reda et al., 2019 (link)).
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