The liver triglycerides (TG) and cholesterol were processed by chloroform-methanol extraction based on a modified Folch method [25 (link)], and measured by an enzymatic reaction with colorimetric assay kits (ThermoFisher Scientific, Mississauga, ON, Canada and Randox Laboratories, Crumlin, UK). The plasma insulin was analysed using an ultrasensitive ELISA kit (Alpco, Salem, NH, USA), in accordance with the manufacturer′s instructions.
Ultrasensitive elisa kit
Manufactured by ALPCO
Sourced in United States
The Ultrasensitive ELISA kit is a laboratory tool designed for highly sensitive detection and measurement of specific analytes in a sample. It utilizes the principles of enzyme-linked immunosorbent assay (ELISA) to provide accurate and reliable results.
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Lab products found in correlation
Colorimetric assay kit, by Thermo Fisher Scientific (1 mentions)
Free glycerol, by Merck Group (1 mentions)
Cholesterol, by Randox (1 mentions)
Quantikine high sensitivity elisa kit, by R&D Systems (1 mentions)
Au5800, by Beckman Coulter (1 mentions)
Advia centaur, by Siemens (1 mentions)
Cobas 6000, by Roche (1 mentions)
Glucose god fs kit, by DiaSys (1 mentions)
Nefa c kit, by Fujifilm (1 mentions)
Tg reagent, by Merck Group (1 mentions)
Free glycerol reagent, by Merck Group (1 mentions)
Accu check, by Roche (1 mentions)
10 protocols using ultrasensitive elisa kit
1
Lipid and Insulin Quantification
2
Comprehensive Metabolic Profiling Protocol
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Blood glucose was measured by glucometer (Accu-Chek). Plasma insulin was measured using ultrasensitive ELISA kit (Alpco, Salem, USA); C-peptide was determined using ELISA kit (Crystal Chem, Elk Grove Village, USA). Plasma triglyceride (TG) (Randox Laboratories, Crumlin, UK), non-esterified fatty-acids (NEFA) (Wako Chemicals, Richmond, VA), cholesterol (Randox Laboratories, Crumlin, UK) and free glycerol levels (Sigma–Aldrich) were enzymatically determined according to the manufacturer's instructions.
3
Biomarker Profiling for Metabolic Health
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Plasma glucose and TG concentrations were measured by using enzymatic and colorimetric methods, respectively (AU5800, Beckman Coulter, California, USA). Serum insulin was measured by using a chemiluminescence immunoassay (ADVIA Centaur, Siemens Healthcare Diagnostics, Hamburg, Germany). These analyses were carried out by a laboratory accredited by the College of American Pathologists. Plasma NEFA was measured at Mayo Medical Laboratories (Rochester, MN, USA) using an enzymatic colorimetric method (Cobas 6000, Roche Diagnostics, Indianapolis, USA).
Plasma interleukin-6 (IL-6) (catalog number HS600B) concentration was measured using Quantikine high-sensitivity ELISA kit (R&D Systems, Minneapolis, MN, USA). Plasma tumor necrosis factor alpha (TNFα) concentration was measured using an ultrasensitive ELISA kit (catalog number 45-TNFHUU-E01, Alpco Diagnostics, Salem, NH, USA). Intra-assay and interassay coefficients of variations for IL-6 and TNFα were <10%.
Plasma total (free and esterified) and urinary free F2-isoprostanes were measured using a method described previously.29 30 (link) The isoprostane levels in plasma and urine were normalized against arachidonic acid and creatinine levels, respectively.
Plasma interleukin-6 (IL-6) (catalog number HS600B) concentration was measured using Quantikine high-sensitivity ELISA kit (R&D Systems, Minneapolis, MN, USA). Plasma tumor necrosis factor alpha (TNFα) concentration was measured using an ultrasensitive ELISA kit (catalog number 45-TNFHUU-E01, Alpco Diagnostics, Salem, NH, USA). Intra-assay and interassay coefficients of variations for IL-6 and TNFα were <10%.
Plasma total (free and esterified) and urinary free F2-isoprostanes were measured using a method described previously.29 30 (link) The isoprostane levels in plasma and urine were normalized against arachidonic acid and creatinine levels, respectively.
4
Characterizing Tpcn2 Knockout Mice
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Wild-type and knockout mice were created using embryonic stem cells from the 129P2 strain carrying a gene trap vector and injected into C57BL/6 blastocysts, as previously described (Calcraft et al. 2009 (link)). We confirmed that Tpcn2 is not transcribed in the knockout mice by running qPCR on multiple tissues in knockout and wild-type mice (see Figure S1 ). PCR was conducted as described above, using primers for GAPDH (forward 3′-CTGAAGGGCATCTTGGGCTA-5′ and reverse 3′-GCCGTATTCATTGTCATACCA-5′) and Tpcn2 (forward 3′-CCAGGCTACCTGTCCTACCA-5′ and reverse 3′-CAGGAAGCGAAACACAATCA-5′). Heterozygous mice were bred and set up as breeder pairs. Male Tpcn2 knockout, heterozygote, and wild-type mice from the heterozygote breeders were phenotyped at 9–13 weeks of age (n = 5–7 in each group), using the IPGTT, described above and in Solberg Woods et al. (2010 (link), 2012) (link). Blood was collected after an overnight fast and at 15, 30, 60, 90, and 120 min after a 1-g/kg glucose injection. We used the Ascensia Elite system for reading blood glucose values (Bayer, Elkhart, IN). We also collected blood at each time point for subsequent analysis of plasma insulin levels, which was assayed using an ultrasensitive ELISA kit from Alpco Diagnostics (Salem, NH).
5
Maternal-Fetal Biochemical Profile
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Biochemical variables in mother and umbilical cord blood (glucose and lipid profile) were measured by colorimetric enzymatic methodology (Spinreact). Maternal HbA1c detection was performed by using a cation exchange resin kit (Eagle diagnostics). Neonatal and maternal insulin concentrations were measured using an ultra-sensitive ELISA Kit (ALPCO). The HOMA IR Index was calculated by the following formula: Fasting insulin (μU/ml) × fasting glucose (mmol/L)/22.5.
6
Retro-orbital Blood Sampling for Metabolic Assays
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Blood was collected in EDTA tubes through retro-orbital sampling. Except for tolerance tests (see above), blood was collected around 8 am. Mouse plasma glycerol was measured by enzymatic assay (free glycerol reagent, Sigma-Aldrich); plasma nonesterified fatty acid and triglyceride levels were measured using the NEFA C kit (Wako) and TG reagent (Sigma-Aldrich), respectively. Glucose levels were measured using a glucometer (AccuCheck, Roche) or Glucose GOD FS kit (DiaSys). Plasma insulin was measured using an ultrasensitive ELISA kit (ALPCO Diagnostics).
7
Insulin Resistance and Glucose Tolerance in Mice
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At week 17, mice were fasted for 6 h and insulin tolerance tests (ITT) were performed after intraperitoneal injections of insulin (0.75 UI/kg body weight). Glycemia was measured with an Accu-Check glucometer (Bayer) before (0 min) and after (10, 20, 30, 60, and 90 min) insulin injection. At the end of week 19, mice were fasted overnight (12 h) and oral glucose tolerance tests were carried out (OGTT, 1 g of glucose/kg body weight). Blood was collected before (0 min) and after (15, 30, 60, 90, and 120 min) glucose challenge for glycemia determination. Blood samples (∼30 μL) were collected at each time point during OGTT and insulinemia was determined using an ultra-sensitive ELISA kit (Alpco, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated based on the following formula: fasting insulinemia (μUI/mL) x fasting glycemia (mM)/22.5.
8
Maternal and Neonatal Metabolic Biomarkers
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Glucose, triglycerides, and cholesterol concentrations (total, HDL, LDL and VLDL) in both maternal and neonatal serum were measured by colorimetric enzymatic assays (Spinreact) following manufacturer's instructions. HbA1c was measured from the maternal plasma by chromatography with Labona Check equipment and reagents. Insulin concentration in maternal and neonatal serum was measured using an ultrasensitive ELISA kit (ALPCO). HOMA IR index was calculated using the following formula: Fasting insulin (μU/mL) × fasting glucose (mmol/L)/22.5. IGF1, IGF2, IGFBP1 and IGFBP3 serum concentrations were measured using ELISA kits (ALPCO). Total adiponectin in maternal serum was measured by ELISA (MyBioSource). Leptin in maternal and umbilical cord blood was measured using the human Leptin immunoassay (Quantikine, R&D Systems) ELISA kit. Maternal serum Acyl (MyBioSource) and Desacylghrelin (SPI-BIO) were measured by ELISA following the manufacturer's instructions.
9
Oral Glucose Tolerance Test in Mice
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Oral glucose tolerance test was conducted on mice fasted for 6 h before the test. An oral gavage of glucose (25%, dissolved in saline) was administered to mice (at 2 g/kg body weight) and blood glucose was measured at 0 min (before gavage), 15 min, 30 min, 60 min and 120 min post gavage using glucose test strips and TRUE result glucometer (Trividia Health; Ft. Lauderdale, FL). Insulin levels in plasma were determined by using the ultrasensitive ELISA kit (Alpco; Salem, NH) according to manufacturer’s instructions. ELISA plate readings of OD450 nm were obtained using a Spectramax M2 plate reader, running Softmax Pro 5.4.3 and standardized to a 5-parameter logistic curve.
10
Oral Glucose Tolerance Test in Rats
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At week 19, rats were fasted overnight (12 h) and oral glucose tolerance tests (OGTT, 2 g of 50% glucose/kg body weight) were carried out as described by Wang et al. [29 (link)] with minor modification. Blood samples were collected through the tail vein, and levels of blood glucose were determined by ONETOUCH UltraEasy glucometer (Johnson, USA) before (0 min) and after glucose injection at different time points (15, 30, 60, and 120 min). The total area under the curve (AUC) was calculated for OGTT.
Blood samples were collected from the visual abdominal aorta at the end of the study. The fasting glycemia and insulinemia were determined using an ultra-sensitive ELISA kit (Alpco, USA). The homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated based on the following formula: fasting insulinemia (mUI/mL) × fasting glycemia (mM)/22.5.
Blood samples were collected from the visual abdominal aorta at the end of the study. The fasting glycemia and insulinemia were determined using an ultra-sensitive ELISA kit (Alpco, USA). The homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated based on the following formula: fasting insulinemia (mUI/mL) × fasting glycemia (mM)/22.5.
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