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Human insulin elisa kit

Manufactured by ALPCO
Sourced in United States

The Human Insulin ELISA Kit is a quantitative enzyme-linked immunosorbent assay (ELISA) for the measurement of human insulin levels in biological samples. The kit utilizes a monoclonal antibody specific for human insulin that is coated on a microplate. Samples and standards are pipetted into the wells and any insulin present is bound by the immobilized antibody. After washing, an enzyme-linked polyclonal antibody specific for insulin is added and binds to the captured insulin. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added and color develops in proportion to the amount of insulin bound. The intensity of the color is measured using a microplate reader.

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31 protocols using human insulin elisa kit

1

Glucose-Stimulated Insulin Secretion Assay

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The protocol was adapted from a previous study [31 (link)]. For GSIS in vitro, SC-islets needed to be fasted in Krebs-Ringer buffer [33 (link)] supplemented with 2 mM glucose for 2 h in a 37 °C, 5% CO2 incubator. Then the SC-islets were stimulated alternately by Krebs-Ringer buffer with low (2 mM) or high (20 mM) glucose. Supernatants were collected after 30 min of each stimulation and the pellets were lysed overnight in acidified alcohol (75% alcohol, 1.5% HCl) at −20 °C, for insulin content measurement. Secreted insulin or total insulin content was measured by a human insulin ELISA kit (ALPCO).
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2

FAHFA Biosynthesis and Bioassays

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The following FAHFAs were obtained from Cayman Chemical (Ann Harbor, MI): 5-PAHSA (Cat# 17043), 9-PAHSA (Cat# 17037), 10-PAHSA (Cat# 19973), 12-PAHSA (Cat# 17107), 13-PAHSA (Cat# 17044), 5-POHSA (Cat# 17114), 9-POHSA (Cat# 17040), 10-POHSA (Cat# 19974), 12-POHSA (Cat# 17105), 13-POHSA (Cat# 17111), 5-OAHSA (Cat# 17115), 9-OAHSA (Cat# 17039), 10-OAHSA (Cat# 19975), 12-OAHSA (Cat# 17108), 13-OAHSA (Cat# 17112), 5-SAHSA (Cat# 17113), 9-SAHSA (Cat# 17109), 10-SAHSA (Cat# 19976), 12-SAHSA (Cat# 17106), and 13-SAHSA (Cat# 17110). R and S enantiomers of 9-PAHSA were synthesized as previously described (25 (link)). Concentrations of FAHFAs in all biological assays is 20 μM, concentration of DMSO is 0.01% unless mentioned otherwise. The Human Insulin ELISA kit (Cat#80-INSHU-E01.1) was purchased from ALPCO (Salem, NH). The Mouse Insulin ELISA kit (Cat# 90080) was obtained from Crystal Chem (Elk Grove Village, IL). Mouse Tnfα (Cat# 430901) and Il-6 (Cat# 431301) ELISA kits were obtained from BioLegend (San Diego, CA). [3H]Deoxy-glucose (NET328A) was obtained from PerkinElmer (Waltham, MA). All the other chemicals were purchased from Sigma-Aldrich (St Louis, MO) unless otherwise stated.
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3

Quantitative Platelet and Pancreatic Assays

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Purified GP2B3A complex (MBS135714), mouse rCela2a protein (MBS1246487), human rCELA2A (MBS1090462) and the CELA2A ELISA kit (MBS932150) were purchased from MyBiosource. CELA2A antibody (SAB1104798) and the elastase substrate N-Succinyl-Ala-Ala-Pro-Leu p-nitroanilide substrate (S8511) were purchased from Sigma-Aldrich. The FITC-conjugated PAC1 antibody (340507, BD Biosciences) and tetramethylrhodamine ethyl ester (TMRE) reagent (T669, Thermo Fisher Scientific), Annexin V-FITC (640905, Biolegend), CD41-FITC (303703, Biolegend) were used for platelet aggregation analysis. Total AKT (CST, 9272), pAKT (S473) (CST, 9271s), pAKT (T308) (CST, 9275s), total S6k (CST, 9202), pS6k (T389) (CST, 9206), total IRS (CST, 2382), pIRS (Y608), (CST, 2385S), pIRS (S636/S639) (CST, 2388s), Gapdh (CST, 3683s), and Actin (CST, 4970) antibodies were purchased from Cell Signaling Technologies. Recombinant A1AT (alpha1 antitrypsin, ab91136), A1AT antibody (ab9400) and ITGA2B antibodies (ab63983) were purchased from Abcam. Human c-peptide ELISA kit (80-CPTHU-E01.1, ALPCO), Human Glucagon ELISA kit (10-1271-01, Mercodia), Rat c-peptide ELISA (80-CPTRT-E01, ALPCO), Mouse Ultrasensitive Insulin ELISA (80-INSMSU-E01, ALPCO), Human Insulin ELISA kit (21-IAAHU-E01, ALPCO), Rat Insulin ELISA kit (80-INSRT-E01, ALPCO) was applied for C-peptide, glucagon, and insulin measurements.
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4

Insulin Secretion in INS-1E Cells

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INS-1E cells were pre-incubated for 2 h in Krebs Ringer buffer (KRB) [62 (link)] containing 1.4 mM glucose, followed by a 1 h incubation at 1.4 or 16.7 mM glucose. Supernatant from the incubation buffers was collected and cleared by centrifugation. Insulin content extraction was performed using acid ethanol. Insulin release and contents were measured by homogenous time-resolved fluorescence (HTRF) (Cisbio bioassays, Codolet, France) according to the manufacturer’s instructions. HTRF signals were measured using Pherastar FS (BMG Labtech, Ortenberg, Germany) microplate reader. Insulin release was normalized to insulin or DNA content (as indicated). To measure static glucose-stimulated insulin secretion, human islets were incubated first in KRB buffer supplemented with 4 mM glucose for 30 min followed by exposure to KRB buffer containing 16 mM glucose for 30 min. Insulin was measured by using a human insulin ELISA kit (ALPCO). Insulin values were normalized to insulin content.
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5

Fasting Blood Lipid and Glucose Profile

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After 12 hours of fasting, 8ml of venous blood from the brachial vein of the participants was obtained to measure the level of blood lipids and blood sugar. Serum was separated by centrifugation with Green Vac-Tube (3000rpm, 15 minutes), which contained a gel-type coagulation activator. The serum was then stored frozen in a -80 ℃ ultra low temperature freezer. The stored serum was used for the analysis of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and fasting blood glucose (FBG) using Vitro Chemistry DT60Ⅱ (Johnson & Johnson, NY, USA). Insulin was measured using a Human Insulin ELISA kit (ALPCO Diagnostics) (the CVs for intra-and interassays = 4.3% and 6.8%, respectively). HOMA-IR (Homeostasis model assessment of insulin resistance) was calculated by [fasting insulin(μU/mL) × fasting glucose (mM)/22.5] as proposed by Matthews, etc. [26 (link)].
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6

Insulin Secretion Assessment in IPCs

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To measure insulin secretion, IPCs (Day 21) were sampled. Clusters were washed with Krebs-Ringer bicarbonate HEPES buffer (KRBH; 116 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2 1.2 mM KH2PO4, 1.2 mM MgSO4, 24 mM HEPES, 25 mM NaHCO3, and 0.1% BSA) and were then preincubated in KRBH buffer for 1 h. The cells were then incubated with KRBH buffer containing 2.5 mM glucose at 37 °C for 60 min. The respective conditioned supernatant was collected and analyzed. Clusters were lysed with RIPA lysis buffer, and protein content was measured with a BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). The collected supernatant was used for insulin analysis with a human insulin ELISA kit (STELLUX, Alpco, Salem, NH, USA). The amount of insulin was normalized to the amount of total protein in the corresponding cell lysate. The representative data were chosen from 5 independent experiments performed in total and are shown in Figures 1 and 5.
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7

Intranasal Human Insulin Pharmacokinetics

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Human insulin (HumulinR, 100 IU/ml; Eli Lilly, Toronto, Ontario, Canada) was administered intranasal at 2.4 IU/mouse as described [26 (link)]. For pharmacokinetics measurements, insulin was administered to three to eight mice per group, animals were sacrificed at the indicated time points (Fig. 1) for isolation of plasma, cerebrospinal fluid (CSF), and cortex. Insulin concentrations were determined by human insulin ELISA kit (Alpco, Salem, New Hampshire, USA) [26 (link)].
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8

Plasma Metabolite Analysis by LC-MS

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Plasma samples were deproteinized using dry sulfosalicylic acid, and frozen at −80°C until analysis. Enrichment analysis was performed by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) (QTrap 5500 MS; AB Sciex) with ExpressHT Ultra LC (Eksigent Div.; AB Sciex) after derivatization with 9-fluoren-9-ylmethoxycarbonyl (Fmoc) [23 ]. Ions of mass to charge ratios of 234, 235, and 239 for phenylalanine and 466, 467, 468, and 470 were utilized. Plasma insulin concentrations were measured by using commercially available human insulin ELISA kit (Alpco Diagnostics, Salem, MA). Plasma human growth hormone concentrations were measured using a commercially available ELISA kit (Invitrogen; Thermo Fisher Scientific, Carlsbad, CA). Plasma amino acid concentrations were measured using liquid chromatography-mass spectrometry (QTrap 5500 MS; AB Sciex) using the internal standard method [24 (link)].
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9

Glucose-Stimulated Insulin Secretion Assay

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An equal number of islet cells, mixed or treated islet-MSC cells, INS1-E and mCherry INS1-E cells were used to perform glucose-stimulated insulin secretion as described before [61 (link)]. Cells were pre-incubated in Krebs-Ringer bicarbonate buffer (KRB) including 2.8 mM glucose for 30 min which was replaced by 2.8 mM glucose KRB for 1 h (basal), followed by an additional 1 h incubation in 16.7 mM glucose KRB (stimulated insulin secretion). Cells were lysed in lysis buffer (20 mM Tris acetate, 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% Triton X-100, 5 mM sodium pyrophosphate, 10 mM β-glycerophosphate plus protease and phosphatase inhibitors; Pierce) and secreted insulin and insulin content from lysed measured by mouse (for rat INS-1E cells; cross reactivity is 100%) or human insulin ELISA kit (for human islets; ALPCO Diagnostics, Salem, NH, USA). Insulin content was normalized to total protein measured by BCA protein assay kit (Pierce, Rockford, IL, USA) as described before [62 (link)].
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10

Biochemical and Hormone Profile Analysis

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For biochemical and hormone profile analysis, fresh, whole-blood samples (20 mL) were collected from each participant in the morning hours, after overnight fasting. Plasma was isolated using ethylenediaminetetraacetic acid (EDTA). For serum isolation, whole blood was previously allowed to clot at room temperature for 20 min. Whole-blood samples were centrifuged at 3000 rpm for a total of 10 min at a temperature of 4 °C.
Immunoassay was performed via chemiluminescent detection for vitamin B12, total 25(OH)D3, insulin, sex hormone-binding globulin (SHBG), and dehydroepiandrosterone sulfate (DHEA-S). Insulin levels were assessed using a Human Insulin ELISA kit (ALPCO Diagnostics) with inter- and intra-assay precision below 15%.
The enzymatic method was used to assess blood glucose levels using an Abbot ALinity I analyzer. Ca, P, and ALP concentrations were estimated using Abbot ALinity C Analyzer (Abbott, Abbott Park, Chicago, IL, USA). Androstenedione was analyzed using an Immulite Siemens analyzer (Siemens Healthcare GmbH, Erlangen, Germany).
HbA1c (%) was assessed with a Siemens DCA Vantage analyzer (Siemens Healthcare GmbH, Erlangen, Germany).
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