Supernatants from cell cultures were analyzed for IFNγ and IL-2 by ELISA according to manufacturer’s instructions (BD Biosciences). ELISAs were read on a SpectraMax M2 microplate reader (Molecular Devices), and data were analyzed using SoftMax Pro version 5.4.2 software (Molecular Devices). Lactate, a byproduct of aerobic glycolysis, was measured in culture supernatants using the Accutrend Plus meter and lactate strips (Roche) [44 (link), 45 (link)]. Samples with high concentrations of lactate were diluted 1:2 in dI H20 to obtain a reading within the meter’s range.
Accutrend plus meter
Manufactured by Roche
Sourced in Germany, United Kingdom
The Accutrend Plus meter is a portable, battery-powered device designed for the quantitative measurement of various analytes in small sample volumes. It provides rapid and accurate results for parameters such as glucose, lactate, and cholesterol levels. The device is intended for professional use in clinical and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Nucleocounter nc 3000, by ChemoMetec (2 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
Softmax pro, by Molecular Devices (1 mentions)
β hydroxybutyrate assay kit, by Merck Group (1 mentions)
Alt activity assay kit, by Merck Group (1 mentions)
Bm lactate test strips, by Roche (1 mentions)
Acetoacetate colorimetric assay kit, by Abcam (1 mentions)
Glucagon elisa kit, by Mercodia (1 mentions)
Accu check advantage, by Roche (1 mentions)
Ultrasensitive mouse insulin elisa, by Mercodia (1 mentions)
Braun thermoscan pro 6000, by Hill-Rom (1 mentions)
Bp 200 plus, by Schiller (1 mentions)
Pronto 7, by Masimo (1 mentions)
Reagent a100, by ChemoMetec (1 mentions)
Reagent b, by ChemoMetec (1 mentions)
Wistar rat, by Charles River Laboratories (1 mentions)
11 protocols using accutrend plus meter
1
Cytokine and Lactate Analysis in Cell Cultures
2
Metabolic Biomarker Analysis in Liver Samples
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Serum glucose and triglyceride levels were measured using the Accutrend Plus meter (Roche Diagnostics, Indianapolis, IN). Serum β-hydroxybutyrate and liver ALT levels were measured using the β-hydroxybutyrate Assay Kit and ALT Activity Assay Kit, respectively (Sigma-Aldrich, Saint Louis, MO). Lipids were extracted from the liver samples by the Bligh/Dyer method and analyzed by gas-liquid chromatography as previously described (Zadravec et al., 2010 (link)).
3
Glucose and Lactate Production in Cells
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After transfection, cells were trypsinized and seeded into 6-well plates to allow the cells to form a complete monolayer. Glucose production was measured by the Contour next blood glucose monitoring system (meter, test strips and control solution). The Contour next system is intended for the quantitative measurement of glucose (from 0.6 mmol/L to 33.3 mmol/L) using approximately 10 μL of the supernatant (1 drop). Lactate production was measured by the Accutrend BM-lactate test strips with the Accutrend Plus meter from Roche (Art. No 128633, Basel, Switzerland), using approximately 10 μL of the supernatant (1 drop). The measurement took place at 24 h, 48 h, and 72 h without changing the medium.
4
Metabolic Biomarker Profiling in Fasted Mice
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Blood samples (4 µL) were collected from the tail at fed condition and after 16.5 hours of fasting. Blood glucose and triglyceride levels were determined using Accu-Check Advantage glucometer and Accutrend Plus meter, respectively (Roche, Germany). Blood ketone levels were measured using Freestyle Optimum ketone meter (Abbott Laboratories, US). Serum insulin, glucagon, and acetoacetate levels were determined in blood samples collected at sacrifice. Blood samples were collected in micro-collection tubes (BD, US) with serum separator additive and left to clot for 30 min at room temperature. Thereafter, serum was collected after centrifugation at 15,000 g for 2 min. Serum insulin and glucagon levels were determined using ultrasensitive mouse insulin ELISA and glucagon ELISA kits, respectively (Mercodia, Sweden). Serum acetoacetate levels were determined using acetoacetate colorimetric assay kit (BioVision, Inc., US). Serum lithium levels were determined using lithium assay kit #LI01ME (Metallogenics, Japan), in serum collected at 90 minutes after [3-13C]acetoacetate injection.
5
Vital Signs Measurement at Rest
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Each participant was asked to sit on a chair, roll up one sleeve, and relax for five minutes, so that the measurements were made at rest. Subsequently, they underwent an electrocardiogram and spirometry to verify normal results. A second stage consisted of serial measurement of vital signs, including heart rate, systolic blood pressure, diastolic blood pressure, tympanic temperature, total hemoglobin, perfusion index, and baseline lactic acid levels.
Blood pressure and heart rate were measured with the use of oscillometric sphygmomanometry (Schiller BP 200 Plus; Schiller, Baar, Switzerland). For measuring tympanic temperature we used a Braun Thermoscan Pro 6000 (Welch Allyn, Inc., New York, USA) with ExacTemp™ technology. Total hemoglobin values and perfusion index were monitored with a multiparameter monitor Pronto 7 (Masimo, California, USA), software version b99e80000004ef796 (2.2.15), and sensor revised version a83f90f0000c53f2. For the determination of lactic acid levels we used an Accutrend Plus meter (Roche Diagnostics, Mannheim, Germany), with a measurement range of 0.8-21.7 mmol/L.
Blood pressure and heart rate were measured with the use of oscillometric sphygmomanometry (Schiller BP 200 Plus; Schiller, Baar, Switzerland). For measuring tympanic temperature we used a Braun Thermoscan Pro 6000 (Welch Allyn, Inc., New York, USA) with ExacTemp™ technology. Total hemoglobin values and perfusion index were monitored with a multiparameter monitor Pronto 7 (Masimo, California, USA), software version b99e80000004ef796 (2.2.15), and sensor revised version a83f90f0000c53f2. For the determination of lactic acid levels we used an Accutrend Plus meter (Roche Diagnostics, Mannheim, Germany), with a measurement range of 0.8-21.7 mmol/L.
6
Microcarrier-based Cell Culture Characterization
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Daily imaging was done by phase contrast microscopy. Cell counts and viability were performed using the Nucleocounter NC3000 (Chemometec) as per manufacturer's instructions for two separate samples. Counts were performed directly on the microcarriers using the reagent A100 and reagent B protocol. Briefly, the cell‐microcarrier suspension was diluted to a 1:1:1 ratio with reagent A100 and reagent B (Chemometec); reagent A100 lyses the cells from the microcarriers releasing the nuclei, while reagent B stabilizes the suspension. The resulting suspension was loaded onto a Nucleocassette Via‐1, preloaded with acridine orange and DAPI and the cassette then transferred Nucleocounter NC3000 machine for processing.
Spent medium samples were collected before and after medium exchanges in the bioreactor and were analyzed for glucose and lactate concentrations on an AccuTrend Plus meter (Roche). Fresh growth medium was used as baseline control.
Based on cell counts, the following parameters were calculated:
Spent medium samples were collected before and after medium exchanges in the bioreactor and were analyzed for glucose and lactate concentrations on an AccuTrend Plus meter (Roche). Fresh growth medium was used as baseline control.
Based on cell counts, the following parameters were calculated:
Specific growth rate
Doubling time
Fold increase
7
Postprandial Triglyceride Dynamics
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At week 15, fasting triglyceride (TG) levels (0 min) were measured from tail vein blood after 14 h of fasting. Then, olive oil (2 mL/kg body weight) was administered orally, and TG measurements were taken from tail vein blood samples taken at 120, 180, 240, and 360 min time points after fat administration. The TG measurements were performed using an Accutrend Plus meter (Roche, Brighton, MA, USA). The TG AUC in the OFTT was calculated from measurements taken before (0 min) and after (up to 360 min) fat administration.
8
Hemorrhagic Shock and Resuscitation in Rats
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HS was performed as previously described 8, 34 . Briefly, rats were anesthetized with sodium thiopentone (120 mg/kg i.p. maintained using 10 mg/kg i.v.). Blood was withdrawn from the right carotid artery and collected with 2 IU/ml of heparin until the mean arterial pressure (MAP) reach 30±2 mmHg, which was maintained for 1.5 h. The shed blood was kept between 6-10 o C. After 1.5h of initiation of hemorrhage, resuscitation was performed with the shed blood over a period of 5 min. An infusion of Ringer Lactate (1.5 mL/kg/hour; i.v.) was maintained throughout the experiment for a total of 4h. The last 3h urine was obtained for the estimation of creatinine clearance. Four hours after resuscitation, blood was collected from carotid artery for measurement of lactate (Accutrend Plus Meter, Roche Diagnostics, UK) and organ injury parameters (IDEXX Ltd, UK), and tissue samples were taken, placed on liquid nitrogen and stored at -80 o C. Sham-rats were used as control and underwent identical surgical procedures, but without hemorrhage.
9
Metabolite Analysis of 3D Cultures
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Supernatants collected from 3D culture samples were analyzed for glucose and lactate concentrations on an Accutrend Plus meter (Roche, Basel, Switzerland) according to manufacturer's instructions. Fresh DMEM-HG supplemented with 10% hSerB was used as baseline control for both metabolites.
10
Cell Growth and Viability Analysis on Microcarriers
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Imaging was performed by either phase-contrast microscopy or fluorescence microscopy. Live/Dead Staining (calcein-AM/ethidium Homodimer) Kit (Thermo Fisher Scientific) was used to assess cell viability on microcarriers following manufacturer's instructions. Cell counts were performed using the NucleoCounter NC-3000 (Che-moMetec). For the spinner flasks cultures, cell counts were performed directly onto microcarriers using the reagent A100 and reagent B protocol. Briefly, the cell-microcarrier suspension was diluted in a 1:3 ratio with reagent A100 (lysing agent) and then reagent B (stabilizing agent). The resulting suspension was then loaded onto a NucleoCassette that contains acridine orange and DAPI. Spent medium samples were collected before and after medium exchanges and analyzed for glucose and lactate concentrations on a Roche Accutrend Plus meter. Fresh growth medium was used as baseline control. On the basis of cell counts, the following parameters were calculated:
1. Specific growth rate
2. doubling time
where µ is the specific growth rate (h -1 ); t d is the doubling time (hr); FI is the fold increase; CPDL is a cumulative population doubling; Cx (t) and Cx(0) represent cell numbers at the end and the start of the culture; and t represents time in culture (hr).
1. Specific growth rate
2. doubling time
where µ is the specific growth rate (h -1 ); t d is the doubling time (hr); FI is the fold increase; CPDL is a cumulative population doubling; Cx (t) and Cx(0) represent cell numbers at the end and the start of the culture; and t represents time in culture (hr).
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