Assessments of glucose homeostasis were performed according to recommendations from the Mouse Metabolic Phenotyping Consortium.39 (link) Glucose tolerance tests were performed after a 6 h morning fast. Mice received a 1 g/kg dose of glucose by intraperitoneal injection and blood samples were collected at set time points by tail massage to determine plasma glucose levels. Hyperinsulinemic euglycemic clamps were performed as previously described with minor modifications.40 (link) Five days prior to study, an indwelling catheter was surgically placed in the right jugular vein and passed subcutaneously such that it could be concealed in the scapular region on the dorsal side of the mouse. Mice were fasted overnight prior to study. Fasted and insulin-stimulated rates of glucose turnover were measured by isotope dilution using a primed/continuous infusion of 3-3H-glucose (Perkin Elmer NET331C001MC; prime = 0.7 μCi/kg over 3 min; 0.05 μCi/min fasted, 0.1 μCi/min clamp). Insulin was provided as a primed/continuous infusion (Novolin-R, Novo Nordisk; prime = 30 mU/kg over 3 min; continuous = 4.5 mU × kg−1 × min−1). Plasma glucose levels were checked every 10 min during the hyperinsulinemic infusion by tail vein message to collect blood and euglycemia was matched between groups using a variable infusion of 20% dextrose (Sigma Aldrich D9434).
D9434
Manufactured by Merck Group
Sourced in United States
The D9434 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose instrument used in scientific research and analysis. The core function of the D9434 is to facilitate the measurement and examination of various samples and materials within a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Novolin r, by Novo Nordisk (1 mentions)
S8636, by Merck Group (1 mentions)
Cary 5000, by Agilent Technologies (1 mentions)
Accu chek glucometer, by Roche (1 mentions)
I0908, by Merck Group (1 mentions)
Breeze2, by Bayer (1 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
Gedatolisib, by Pfizer (1 mentions)
S5886 1kg, by Merck Group (1 mentions)
10 protocols using d9434
1
Glucose Homeostasis Assessment in Mice
2
Glucose Tolerance and Insulin Response in Mice
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For the GTT, after 16–18 h of starvation in both groups, mice were injected intraperitoneally with glucose (2 g/kg body wt; Sigma-Aldrich, D9434), and blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min. One week after the GTT measurement, insulin (0.75 unit/kg body wt; Sigma-Aldrich, 1342106) was injected intraperitoneally following starvation for 4 h, and the blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after the insulin injection.
3
Postnatal Neurometabolic Interventions
4
Biofilm Inhibition Assay of Compounds
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Staphylococcus epidermidis (ATCC 35984) grown in Tryptic Soy Broth (TSB, 105459, Merck, Kenilworth, NJ, USA) overnight at 37 °C was diluted in fresh medium with 1% glucose (D9434, Sigma-Aldrich) before being transferred to a 96-well microtiter plate; 50 µL/well were incubated overnight with 50 µL of compound 1 –6 dissolved in Milli-Q water added in duplicates. The bacteria were then removed from the plate and the plate washed with tap water. The biofilm was fixed at 65 °C for 1 h before 70 µL 0.1% crystal violet (115940, Merck Millipore) was added to the wells for 10 min of incubation. Excess crystal violet solution was then removed and the plate dried for 1 h at 65 °C. Seventy microliters of 70% EtOH were then added to each well and the plate incubated on a shaker for 5–10 min. Biofilm formation inhibition were assessed by the presence of violet color and was measured at 600 nm absorbance using a 1420 Multilabel Counter VICTOR3TM. Fifty microliters of a non-biofilm forming Staphylococcus haemolyticus (clinical isolate 8-7A, University hospital, UNN, Tromsø, Norway) mixed in 50 µL autoclaved Milli-Q water was used as a control; 50 µL S. epidermidis mixed in 50 µL autoclaved Milli-Q water was used as the control for biofilm formation; and 50 µL TSB with 50 µL autoclaved Milli-Q water was used as a medium blank control.
5
Glucose Concentration Absorbance Spectra
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A UV–Vis–NIR spectrophotometer (Cary 5000, Varian, Australia) was used to obtain the absorbance spectra of glucose solutions at various concentrations. The highest concentration of the glucose solutions was 9,333 mg/dL, achieved by dissolving dextrose powder (D9434, Sigma-Aldrich, USA) in heavy water, and was diluted to various concentrations down to 1% (93 mg/dL). The absorbance spectrum of heavy water, denoted by 0 mg/dL, was also obtained as a control. Each sample was separately placed in a quartz cuvette with a 1 cm light path ( ), and their transmittance was measured over a wavelength range of 500–2,000 nm, with a 1 nm step size. The absorbance ( ) was converted from the measured percent transmittance ( ) using the equation , and absorption coefficient can be calculated as .
6
Intraperitoneal Glucose Tolerance Test in Mice
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An ACCU-CHEK glucometer (Roche Diagnostics, Shanghai, China) was used to test the plasma glucose. An intraperitoneal glucose tolerance test (IGTT) was performed on GPRC6A−/− and GPRC6A+/+ mice aged 8 weeks and 12 months according to a published experimental method [31 (link)]. Briefly: (1) At 17:00 pm on the first day, WT and KO mice were weighed and then deprived of feed while having access to water. (2) At 9:00 am the next day, tail blood of the mice was collected to measure glucose, which was recorded as the blood glucose as 0 min. (3) The abdomen of mice was sterilized with 70% ethanol, followed by intraperitoneal glucose (Sigma-Aldrich, D9434, Saint Louis, USA) injection (2 mg/kg body weight). (4) Blood glucose was measured at 15, 30, 60, 120 and 180 min. (5) The mice were placed back into the cage and fed with feed.
7
Glucose Homeostasis in Aging Mice
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Insulin tolerance tests (ITT) and glucose tolerance tests (GTT) were performed after 9 months of dietary intervention, when mice were 13 months of age, to examine systemic glucose homeostasis. We also performed ITT and GTT on the 4-month-old young reference group after 2 weeks of AIN-93M diet feeding, which allowed us to assess the effect of age on glucose homeostasis.
Prior to ITT and GTT mice were food-deprived for 4 or 12 h, respectively. Insulin (0.75 mU /g body weight, I0908, Sigma-Aldrich, St. Louis, MO, USA) or glucose (0.4 mg dextrose/g body weight, D9434, Sigma-Aldrich, St. Louis, MO, USA) solutions were administrated through intraperitoneal injection. Tail vein blood glucose levels were determined by glucometer (Breeze2, Bayer, Whippany, NJ, USA) at 0, 30, 60, 90, and 120 min after insulin or glucose administration. Due to the limited blood volume of mice, we did not collect blood samples to measure circulating insulin levels during the tests.
Prior to ITT and GTT mice were food-deprived for 4 or 12 h, respectively. Insulin (0.75 mU /g body weight, I0908, Sigma-Aldrich, St. Louis, MO, USA) or glucose (0.4 mg dextrose/g body weight, D9434, Sigma-Aldrich, St. Louis, MO, USA) solutions were administrated through intraperitoneal injection. Tail vein blood glucose levels were determined by glucometer (Breeze2, Bayer, Whippany, NJ, USA) at 0, 30, 60, 90, and 120 min after insulin or glucose administration. Due to the limited blood volume of mice, we did not collect blood samples to measure circulating insulin levels during the tests.
8
Inhibiting Staphylococcus epidermidis Biofilm
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For testing the inhibition of biofilm formation, the biofilm-producing Staphylococcus epidermidis (ATCC 35984) was grown in Tryptic Soy Broth (TSB, 105459, Merck, Kenilworth, NJ, USA) overnight at 37 °C. The overnight culture was diluted in fresh medium with 1% glucose (D9434, Sigma) before being transferred to a 96-well microtiter plate; 50 µL/well were incubated overnight with 50 µL of the test compound dissolved in 2% (v/v) DMSO aq. added in duplicates. The bacterial culture was removed from the plate and the plate was washed with tap water. The biofilm was fixed at 65 °C for 1 h before 70 µL 0.1% crystal violet (115940, Millipore, Burlington, MA, USA) was added to the wells for 10 min of incubation and 70 µL of 70% ethanol was then added to each well and the plate incubated on a shaker for 5–10 min. Biofilm formation inhibition was assessed by the presence of violet color and measured at 60 nm absorbance using a 1420 Multilabel Counter VICTOR3TM; 50 µL of a non-biofilm forming Staphylococcus haemolyticus (clinical isolate 8-7A, University Hospital of North Norway Tromsø, Norway) mixed in 50 µL autoclaved Milli-Q water was used as a control; 50 µL S. epidermidis mixed in 50 µL autoclaved Milli-Q water was used as the control for biofilm formation; and 50 µL TSB with 50 µL autoclaved Milli-Q water was used as a medium blank control.
9
Gedatolisib Injection Dosage Study in Nude Mice
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8‐ to 9‐week‐old female, Crl:NU(NCr)‐Foxn1nu mice (Charles River, Strain 490, Wilmington, MA, USA) were injected with either vehicle, 1 or 10 mg·kg−1 Gedatolisib (Pfizer, PF‐05212384, New York, NY, USA) via the tail vein. Gedatolisib vehicle consisted of 0.3% l‐lactic acid (Sigma, L6402) and 5% dextrose (Sigma, D9434) in endotoxin‐free ultrapure water (Millipore, TMS‐011‐A, Burlington, MA, USA) and was sterile‐filtered through a 0.2 μm PES filter. Gedatolisib was added to vehicle at 2 mg·mL−1, heated at 60 °C for 15–20 min, and then sterile‐filtered. Sterile PBS was used for balance, for a final injection volume of 100 μL. Animals were euthanized 1 or 2 h following injection.
10
Measuring Cardiac Tissue Contractility via EHT Pacing
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Videos with 5 s duration of paced EHTs were analyzed as previously described [26 (link),27 (link)]. To do so, 24-well metal electrode trays with 2 mL Tyrode solution (1.8 mM calcium chloride (Sigma, C4901, St. Louis, MO, USA), 1.0 mM magnesium chloride (Sigma, 1374248, St. Louis, MO, USA), 5.4 mM potassium chloride (Fisher, P330-500, Hampton, NH, USA), 140 mM sodium chloride (Sigma, S5886-1KG, St. Louis, MO, USA), 0.33 mM monobasic sodium phosphate (Fisher, P284-500, Hampton, NH, USA), 10 mM HEPES (Invitrogen, 15630-080, Waltham, MA, USA), 5 mM dextrose (Sigma, D9434, St. Louis, MO, USA) in H2O) per well were incubated for 30 min at 37 °C, 5% CO2. EHT post arrays were transferred to the electrodes and paced for 5 s with 1.5 Hz, 10 V, 20 ms pulses with 45 fps videos captured using live brightfield microscopy. Maximal twitch force was calculated based on peak length displacement of tissues using previously published MATLAB code.
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