Seahorse xfe96 bioanalyzer

For estimation of the mitochondrial and glycolytic functions of the 2D cultured HconF cells, their oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured, respectively, using a Seahorse XFe96 Bioanalyzer (Agilent Technologies) as basically described in our previous reports [38 (link),46 (link),47 (link)]. Briefly, 20 × 10³ 2D cultured HconF cells were seeded onto the wells of a 96-well Seahorse measurement analytical plate administered without (non-treated control, NT) or with solutions of TGF-β-1, -2, or -3 (5 ng/mL). After exchanging the culture medium with Seahorse XF DMEM assay medium (pH 7.4, Agilent Technologies, #103575-100) supplemented with 5.5 mM glucose, 2.0 mM glutamine, and 1.0 mM sodium pyruvate, the basal OCR and ECAR values were determined using a Seahorse XFe96 Bioanalyzer, and thereafter, the samples were further analyzed after supplementation with 2.0 μM oligomycin, 5.0 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 1.0 μM rotenone and antimycin A, and 10 mM 2-deoxyglucose (2-DG). The OCR and ECAR values were normalized to the amount of protein per well.

The rates of oxygen consumption rate (OCR) and extracellular acidification (ECAR) of 2D HconF cells were measured using Seahorse XFe96 Bioanalyzer (Agilent Technologies) as described previously with minor modifications^{32 (link),33 (link)}. Briefly, 20 × 10³ 2D HconF cells were placed in wells of a 96-well assay plate as follows; (1) non-treated control (NT), (2) treated with TGF-β2, (3) treated with FGF-2 and (4) treated with TGF-β2 and FGF-2. After replacing the culture medium with Seahorse XF DMEM assay medium (pH 7.4, Agilent Technologies, #103,575–100) supplemented with 5.5 mM glucose, 2.0 mM glutamine, and 1.0 mM sodium pyruvate, basal OCR and ECAR values were determined using a Seahorse XFe96 Bioanalyzer and thereafter, the samples were further analyzed after supplementation with 2.0 μM oligomycin, 5.0 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 1.0 μM rotenone and antimycin A, and 10 mM 2-deoxyglucose (2-DG). The OCR and ECAR values were normalized to the amount of protein per well.

As a real-time cellular metabolic function analysis, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of the 2D-cultured HTM cells in the absence or presence of TGF-β2 (5 ng/mL) and/or ATRA (10 μM) were measured using a Seahorse XFe96 Bioanalyzer (Agilent Technologies, Santa Clara, CA, U.S.A.), as described in a recent report [43 (link),44 (link),45 (link)]. Briefly, 20 × 10³ 2D HTM cells under several conditions—(1) nontreated control, (2) treated with TGF-β2, (3) treated with ATRA, and (4) treated with TGF-β2 and ATRA—were grown in 96-well assay plates. After replacing the culture medium with Seahorse XF DMEM assay medium (pH 7.4, Agilent Technologies, #103575-100) supplemented with 5.5 mM glucose, 2.0 mM glutamine, and 1.0 mM sodium pyruvate, the basal OCR and ECAR values were determined using a Seahorse XFe96 Bioanalyzer and the samples were then further analyzed after supplementation with 2.0 μM Oligomycin (Oligo), 5.0 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), 1.0 μM rotenone and antimycin A, and 10 mM 2-deoxy-d-glucose (2-DG). The OCR and ECAR values were normalized to the amounts of proteins per well.

The OCR of islets or NIT-1 cells was analyzed by an XFe96 Seahorse bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. The OCR of islets (15–20 islets/well) was analyzed in triplicate per mouse and normalized by protein content. Briefly, islets were incubated with assay medium (Seahorse XF base medium minimal DMEM, supplemented with 3 mM glucose and 0.2% BSA) for 1 h at 37 °C in a non-CO₂ incubator. Islets were sequentially cultured in high glucose (20 mM), oligomycin (5 μM), FCCP (1 μM), and antimycin/rotenone (5 μM) for 20 min.

The 2D HTM cells untreated or treated with 5 ng/mL TGF-β2 and/or 10 μM BRI were subjected to analyses of their barrier functions using TEER and FITC dextran permeability measurements as described previously [11 (link),16 (link)]. Alternatively, the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of the 2D HTM cells under these conditions were measured as previously described using a XFe96 Seahorse Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) [17 (link)].

Cells were seeded in XFe96 cell culture microplates (Agilent, USA) and treated with compounds for 24 hours. Cells were washed with Seahorse XF DMEM assay buffer (Agilent, USA) supplemented with 10mM glucose, 1mM pyruvate and 2mM glutamine, and incubated for 1 hour at 37°C without CO₂. The ATP production from mitochondrial respiration and glycolytic respiration in response to Oligomycin and Rotenone/Antimycin A was measured using the ATP production assay kit (Agilent, USA). The oxygen consumption rate (OCR) and Extracellular acidification rate (ECAR) from mitochondrial oxidative phosphorylation (OXPHOS) in response to Oligomycin, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and Rotenone/Antimycin A was measured using the mitochondrial stress test kit (Agilent, USA). The glycolytic activity or glycoPER in response to Rotenone/Antimycin A and 2-deoxy-D-glucose was measured using glycolytic rate assay (Agilent, USA). All measurements were performed using Seahorse XFe96 Bioanalyzer (Agilent, USA).

BMDMs were plated at 7 × 10⁴ cells/well into XFe96 cell culture microplates (Agilent) and were allowed to adhere during 4h incubation at 37 °C and 5% CO₂. Next, BMDMs were stimulated with PBS and sEVs from TSP‐pulsed or untreated BMDMs for 24h. Sensor cartridge (Agilent Technology) was pre‐incubated the day before running the XF Assay with 200 μl/well XF96 calibrant solution on one utility plate overnight at 37 °C in a non‐CO₂ humidified incubator. Cell culture media was replaced with RPMI 1640 (GIBCO) supplemented with 1% L‐glutamine + 5% FCS and incubated for 1h at 37 °C without CO2. To measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), different components were prepared, including 10 mM glucose, 1μM Oligomycin, 2μM Carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP), and 1μM Rotenone/Antimycin A (R/AA) with the Seahorse XFe‐96 Bioanalyzer (Agilent) and were analysed using XFe Wave software. Each measurement cycle consisted of 3 min mix, 0 min wait, and 3 min measure. After the measurement, cell numbers were counted and all the results were normalized to the number of cells.