Media and supernatant lactate was measured spectrophotometrically using a Dimension EXL autoanalyser (Siemens, Germany) with Flex© reagent cartridges (product code DF16). In brief, LDH, NAD, dihydrazine sulphate, and tris buffer (40U, 10mmolL-1, 180mmolL-1, and 100mmolL-1 respectively) were added to cell free supernatants. The subsequent exchange of lactate to pyruvate, captured by the hydrazine compound, is directly proportional to the change in NAD+ to reduced NAD+ (NADH) concentration measured at 340-383 nm, from which the initial lactate pool size was inferred. Lactate was not measurable in RPMI and 10% FBS alone. To assess the freeze-thaw stability of lactate 3x106 Pan T cells were cultured in three separate 2 mL wells. All supernatant was removed from the wells at day 2, and three 50 μL fresh supernatant samples from each well were analysed for extracellular lactate. Supernatants were subsequently also taken for analysis after 1, 2, and 3 freeze-thaw cycles, with samples left for at least 24 hours in the freezer before subsequent thawing.
Flex reagent cartridge
Manufactured by Siemens
Sourced in Germany, United States
Flex reagent cartridges are a core component of Siemens' laboratory equipment. They are designed to hold and deliver reagents necessary for various analytical procedures. The cartridges are engineered to ensure accurate and reliable reagent dispensing, supporting the overall functionality of the laboratory equipment.
Automatically generated - may contain errors
Lab products found in correlation
Dimension vista system, by Siemens (4 mentions)
Dimension exl autoanalyser, by Siemens (2 mentions)
Dimension clinical chemistry system, by Siemens (2 mentions)
Dimension exl autoanalyzer, by Siemens (1 mentions)
Dxc 600 analyzer, by Beckman Coulter (1 mentions)
Prorenin, by Innovative Research (1 mentions)
Access ultrasensitive insulin assay, by Beckman Coulter (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Ccl18, by R&D Systems (1 mentions)
Il 1ra, by R&D Systems (1 mentions)
18 protocols using flex reagent cartridge
1
Measuring Lactate in Cell Supernatants
2
Venom-Induced Blood Biochemistry Assessment
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At various time points during the animal experiments (0, 3, 6, 9, and 12 h post-injection of venom or 0.9% NaCl), approximately 0.5 mL of blood was taken via the carotid artery and collected in to 1.5 mL Eppendorf tubes. After collection, the samples were centrifuged at 5,500 rpm for 10 min. The supernatant was stored at –20°C for no longer than 12 h, before determination of creatinine and BUN levels. Creatinine and BUN levels were measured at 37°C via an automated process using Flex reagent cartridges and a Dimension clinical chemistry system supplied by Siemens Healthineers (Germany). Plasma BUN values were measuring using 340 and 383 nm wavelengths by bichromatic rate, whereas plasma creatinine level was measured using 540 and 700 nm wavelengths using bichromatic rate.
3
Quantitative Extracellular Lactate Assay
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Extracellular lactate was measured in cell culture supernatants as a read out of glycolytic activity. Supernatants were collected at various timepoints during expTreg expansion and stored at −20 °C until required. Lactate was measured as previously described68 , briefly: lactate was measured spectrophotometrically using a Dimension EXL autoanalyser (Siemens, Germany) with Flex© reagent cartridges (product code DF16). Lactate dehydrogenase (LDH, 40U), Nicotinamide adenine dinucleotide (NAD, 10 mmolL-1), dihydrazine sulphate (10 mmolL-1) and tris buffer (100 mmolL-1) were added to cell free expTreg expansion supernatants. The subsequent exchange of lactate to pyruvate, captured by the dihydrazine compound, is directly proportional to the change in NAD+ to NADH concentration measured at 340–383 nm, from which the initial lactate pool size was inferred.
4
Lactate Measurement and Freeze-Thaw Stability
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Media and supernatant lactate were measured spectrophotometrically using a Dimension EXL autoanalyzer (Siemens) with Flex reagent cartridges (product code DF16). In brief, lactate dehydrogenase, NAD, dihydrazine sulfate, and Tris buffer (40 U and 10, 180, and 100 mmol/l, respectively) were added to cell-free supernatants. The subsequent exchange of lactate to pyruvate, captured by the hydrazine compound, is directly proportional to the change in NAD+ to NADH+ (NADH) concentration measured at 340–383 nm, from which the initial lactate pool size was inferred. Lactate was not measurable in RPMI 1640 and 10% FBS alone. To assess the freeze–thaw stability of lactate, 3 × 106 Pan T cells were cultured in three separate 2-ml wells. All supernatant was removed from the wells at day 2, and three 50-μl fresh supernatant samples from each well were analyzed for extracellular lactate. Subsequently, supernatants were also collected for analysis after one, two, and three freeze–thaw cycles, with samples left in the freezer for ≥24 h before thawing.
5
Measuring Renal Biomarkers Using Siemens Analyzers
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Urinary and plasma creatinine, albumin, and protein were measured using Siemens Flex reagent cartridges for the respective proteins, while electrolytes were measured using a Siemens V-Lyte integrated multisensor and all read using a Siemens Dimension Vista 1500 chemistry analyzer at Hunter Area Pathology Service (Newcastle, NSW, Australia).
Plasma cystatin C was measured at Pathology New England (Tamworth, NSW, Australia) using a Gentian Cystatin C Immunoassay read on a Beckman Coulter DxC 600 analyzer.
Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure plasma and urinary prorenin (Molecular Innovations, MI), ACE (Duoset, R&D systems, MN), and AGT (IBL International, Hamburg, Germany) according to the manufacturer’s instructions (see also [Sykes et al. 2014a (link),b (link)]). The intra- and interassay coefficient of variation (CV) for each assay was 26.5% and 16.9% (n = 2 plates) for ACE, 7.7% and 7.9% (n = 2 plates) for AGT, respectively. The intra-assay CV for the prorenin assay was 17.1%. The lowest values we could successfully measure for uACE, uAGT, and uprorenin were 0.001 ng/mL, 0.2 ng/mL, and 0.6 pg/mL, respectively.
Plasma cystatin C was measured at Pathology New England (Tamworth, NSW, Australia) using a Gentian Cystatin C Immunoassay read on a Beckman Coulter DxC 600 analyzer.
Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure plasma and urinary prorenin (Molecular Innovations, MI), ACE (Duoset, R&D systems, MN), and AGT (IBL International, Hamburg, Germany) according to the manufacturer’s instructions (see also [Sykes et al. 2014a (link),b (link)]). The intra- and interassay coefficient of variation (CV) for each assay was 26.5% and 16.9% (n = 2 plates) for ACE, 7.7% and 7.9% (n = 2 plates) for AGT, respectively. The intra-assay CV for the prorenin assay was 17.1%. The lowest values we could successfully measure for uACE, uAGT, and uprorenin were 0.001 ng/mL, 0.2 ng/mL, and 0.6 pg/mL, respectively.
6
Neonatal Urine Collection and Analysis
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Twenty-four hour urine samples were collected on days 1–3 of postnatal life as previously described [9] (link). Disposable diapers of the appropriate size containing a pure cellulose pad were used for urine collection. Diapers were changed every 4–6 hours as clinically appropriate and collected in a plastic bag at 4°C until completion of a 24 hr collection period then folded inside out and the urine extracted using a specially constructed press. Each 24 hrs of pooled specimens were stored at −80°C and spun before analysis. Exact 24 hr urinary output was calculated by weighing diapers before and after use. As humidity can contribute to diaper weight, the degree and length of time in humidity were recorded and adjustments were made as appropriate [19] (link). The corrected values were used for 24 hr output values for analysis. Assessment of urinary creatinine was carried out by Hunter Area Pathology Services using the CREA method (a modification of the kinetic Jaffe reaction) with Flex reagent cartridges (Siemens Healthcare Diagnostics Inc., Camberley, United Kingdom) on the Dimension Vista System (Siemens).
7
Metabolic Biomarker Measurement Protocol
8
Measuring Biomarkers in Blood Samples
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Blood samples were drawn into either EDTA containing tubes (purple top) or tubes without any additive. Samples were centrifuged within 30 min at 3,000 g for 10 min and plasma or serum were separated and either analyzed immediately or kept at −80°C. High sensitivity CRP (hsCRP) was measured within 2 to 3 hours after collection using the Flex reagent Cartridge (Date Behring, Newark, DE), which is based on a particle-enhanced turbidimetric immunoassay technique. Serum levels of lipids, including total cholesterol, high-density lipoprotein (HDL) cholesterol, calculated low-density lipoprotein (LDL) cholesterol, and triglycerides, were also assessed with a Flex reagent cartridge (Date Behring, Newark, DE). Plasma insulin levels were measured using a commercially available radioimmunoassay kit (Coat-A-Count Insulin, Cambridge Diagnostic Products, Inc, Fort Lauderdale, FL). Plasma glucose levels were measured using a commercial kit based on the hexokinase-glucose-6-phosphate dehydrogenase method (Flex Reagent Cartridges, Dade Behring, Newark, DE). Insulin resistance was then assessed using the homeostasis model assessment (HOMA-IR) equation (fasting insulin × fasting glucose ÷ 405) (27 (link)). In addition, plasma samples were frozen at −80°C till assay.
9
Biomarker Assays for Diabetes
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Serum fasting blood glucose was assayed on autoanalyzer (Dimension Clinical Chemistry System, Dimension Max. Germany), using Flex reagent cartridges, supplied by Dade Behring, Germany. HbA1c was assayed on autoanalyzer “Hb Gold Analyzer”, using Gold Reagent Kit- HbA1c, provided by Drew Scientific Ltd. Germany. Serum C- peptide was assayed on autoanalyzer by Immulite C-peptide Kit, provided by EURO/DPC Ltd. UK. The antioxidant enzymes and TBARS were assayed on ELx 800 automated microplate reader (BIO-TEK Instrument Inc. USA), using ELISA kits supplied by Cayman Chemical Company, USA.
10
Measuring Biomarkers in Blood Samples
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