7 × 104 INS1 (832/13) or EndoC-βH3 cells were plated in 96 well plate and treated with JQ1 at various concentrations (from 25 nM to 200 nM) for 3 or 4 days [49 (link)]. At the end of the treatment, cells were lysed in lysis buffer (TETG: 20 mM Tris pH 8.0, 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EGTA) and insulin content was measured by insulin ultra-sensitive assay kit (Cisbio). Within an experiment, each point was determined in triplicate and the experiment was repeated at least three times. For RNA extraction, cells were seeded at 5 × 105 (INS1) or 1 × 106 (EndoC-βH3) respectively and treated with various JQ1 concentrations for either 3 or 4 days. Cells were lysed in 1 ml Trizol and total RNAs were extracted according to manufacturer’s instructions.
Insulin ultra sensitive assay kit
Manufactured by PerkinElmer
Sourced in United States, France
The Insulin Ultra-Sensitive Assay kit is a laboratory equipment product designed to measure insulin levels in biological samples. It provides a highly sensitive and accurate method for quantifying insulin concentrations, which is essential for various clinical and research applications.
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Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti p22phox, by Santa Cruz Biotechnology (1 mentions)
Anti p47phox, by Santa Cruz Biotechnology (1 mentions)
Vas 2870, by Enzo Life Sciences (1 mentions)
Goat anti rabbit igg hrp, by Abcam (1 mentions)
Anti cd73, by Santa Cruz Biotechnology (1 mentions)
Gw9508, by Bio-Techne (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Reagents for sds page, by Bio-Rad (1 mentions)
Control sirna, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Linoleic acid, by Merck Group (1 mentions)
Clariostar microplate reader, by BMG Labtech (1 mentions)
Anti α tubulin 3873, by Cell Signaling Technology (1 mentions)
Viacount kit, by Merck Group (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
9 protocols using insulin ultra sensitive assay kit
1
Measuring JQ1 effects on insulin content
2
SDS-PAGE Reagents and Antibodies
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Reagents for SDS-PAGE were obtained from Bio-Rad (Richmond, CA, USA); dihydroethidium (DHE), RPMI 1640 culture medium, heat-inactivated fetal bovine serum, Opti-MEM medium, Lipofectamine 2000, RIPA lysis buffer and Fura-2 AM were obtained from Thermo Fischer Scientific (Waltham, MA, USA); Bradford Reagent and linoleic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA); Insulin Ultra-Sensitive Assay kit was obtained from Cisbio (Codolet, France); GW9508 was obtained from Tocris Bioscience (Bristol, England, UK); VAS2870 was obtained from Enzo Life Sciences (Farmingdale, NY, USA); p22phox siRNA (#sc-61892), control siRNA (#sc-37007), anti-p47phox (#sc-14015; dilution 1:1000 in 5% BSA in TBST), anti-p22phox (#sc-271968; dilution 1:500 in 5% BSA in TBST) and anti-CD73 (#sc-25603; dilution 1:1000 in 5% BSA in TBST) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); goat anti-rabbit IgG HRP (#ab205718; dilution 1:10,000 in 3% skimmed milk in TBST) and goat anti-mouse IgG HRP (#ab97023; dilution 1:10,000 in 3% skimmed milk in TBST) secondary antibodies were obtained from Abcam (Cambridge, UK).
3
Glucose-Stimulated Insulin Secretion in Islets
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After 1 h incubation with different conditions, islets from WT and NOX2 KO mice were checked for GSIS. Groups of 10 islets were collected to fresh tubes containing KH buffer with 0.5 mM glucose and incubated at 37°C for 45 min. Supernatant was discarded and substituted by KH buffer with either 5.6 or 8.3 mM of glucose and incubated at 37°C for 60 min. Supernatant was collected and insulin release was measured by Fluorescence Resonance Energy Transfer (FRET) using the Insulin Ultra-Sensitive Assay kit (Cisbio). The signal intensity was measured at 665 and 620 nm at the CLARIOstar Microplate Reader (BMG LABTECH).
4
Evaluating Cellular Stress Response
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A culture medium with or without phenol red (RPMI 1640); dihydroethidium (DHE); Fura-2 AM and proinflammatory cytokines (IL-1β, TNF and IFN-γ) from Thermo Fisher Scientific (Waltham, MA, USA); collagenase P and 4-phenylbutyric acid (4-PBA) from Sigma-Aldrich (St. Louis, MO, USA); GSK2795039 from Hycultec (Beutelsbach, Germany); a FITC Annexin V Apoptosis Detection Kit with PI from BioLegend (San Diego, CA, USA); a ViaCount kit from Merck Millipore (Burlington, MA, USA); an Insulin Ultra-Sensitive Assay kit from Cisbio (Codolet, France) and anti-p-eIF2-α (#9721S), anti-p-IRE1 and anti-α-tubulin (#3873) antibodies from Cell Signaling Technology (Danvers, MA, USA).
5
Glucose-Stimulated Insulin Secretion Assay
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Mouse islets were isolated as described previously [38 (link)]. For the GSIS assay, batches of five islets were picked with at least four replicates per groups and were preincubated for 1 h in Krebs–Ringer HEPES buffer (KRHB) containing 0.1% (wt/vol.) BSA and 2 mmol/l glucose. Then they were incubated for 1 h at 37°C with KRHB containing either 2 or 20 mmol/l glucose. An aliquot of the buffer was taken, and secreted insulin determined by the insulin RIA kit (Linco/Millipore, Billerica, NJ, USA). The islets were lysed for the measurement of insulin and proinsulin content by the homogeneous time-resolved fluorescence assay using the Insulin Ultra Sensitive Assay Kit (Cisbio, Codolet, France) and the Proinsulin rat/mouse ELISA kit (Mercodia, Uppsala, Sweden), respectively.
6
Insulin Quantification via ELISA Miniaturization
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The secreted insulin was quantified using the Insulin Ultra-sensitive Assay Kit (CISBIO). The ELISA assay was miniaturized from 10 μL to 5 μL (Supplementary Figure 2A ). The ELISA reagent was directly added to the samples at a ratio of 1:1. The sampling plate was incubated overnight at room temperature, and the readout was performed using a microplate reader (Infinite M1000, TECAN, Männedorf, Switzerland).
7
Insulin Secretion Assay for Islets
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Batches of 10 similar-sized islets were selected and checked for the insulin secretion assay, as previously described [23 (link),24 (link)]. Briefly, islets were incubated in KH buffer with 2.8-mM glucose at 37 °C for 30 min. The supernatant was discarded and islets were incubated at 37 °C for 60 min with a KH buffer in a low (5.6 mM) or high (16.7 mM) glucose concentration. The supernatant was collected for insulin measurements. The islets were then pooled for each condition (24 h or 48 h) and disrupted in an acid–ethanol solution (52 mL ethanol : 17 mL water : 1 mL hydrochloric acid) to obtain the intracellular insulin content. Insulin was measured by a Fluorescence Resonance Energy Transfer (FRET) using the Insulin Ultra-Sensitive Assay kit (Cisbio).
8
Perifusion Protocol for Insulin Secretion
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Four slices originating from different embedded blocks were placed into a closed perifusion chamber (Warner Instruments, cat. no. 64-0223 and cat. no. 64-0281 (P-5)) and connected to a perifusion system with automated tray handling (Biorep Technologies, cat. no. PERI4-02-230-FA). In order to equilibrate the slices to 37 C and to wash out accumulated hormones and enzymes from the tissue, a 60-minute flushing step with 3mM KRBH at a flow rate of 100 mL/min was performed. Subsequently, the actual perifusion protocol was applied using a flow rate of 100mL/min and samples were collected in 96-well plates containing aprotinin at a final concentration of 25 KIU/mL at an interval of 2 minutes. After the perifusion experiments, tissue slices were lysed for total insulin content measurements using 500 mL acid ethanol (2% HCl [37%, 12M] in abs. ethanol). Samples were kept at À20 C until measured with an Insulin Ultra Sensitive Assay Kit (Cisbio, cat. no. 62IN2PEH).
9
Glucose-stimulated Insulin Release in Mouse Islets
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For acute insulin release in response to glucose, primary BL6J mouse islets were isolated (21 (link)) and pre-incubated (1 h) in HB-ESC buffer containing 6 mM glucose and pH 7.4 at 37°C. Islets were then incubated in HB-ESC with 6 mM glucose for 5 min (basal) after which the solution was collected and replaced with stimulatory glucose in HB-ESC 8 mM glucose with pH set to 7.4 or 7.1. Solution was collected and replaced sequentially after 5, 10 and 20 min of incubation. Insulin was determined using Insulin Ultra-Sensitive assay kit from Cisbio (Bagnols-sur-Ceze, France). Secreted insulin was normalized to the basal insulin secretion at 6 mM glucose after 5 min and shown as a fold change.
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