Glucose was measured at 2:00 pm at the end of each dietary period following a 5 h fast. For intraperitoneal glucose tolerance tests (IPGTT), blood samples were drawn from the tail vein at 0, 10, 20, 30, 60, 90, and 120 minutes after i.p. injection of 2 g/kg dextrose. Blood glucose was determined using a One Touch II glucose monitor (LifeScan Inc., Milpitas, CA, USA). Fasting insulin was measured in plasma obtained from 50–100 μl of blood collected from tail vein before glucose injection (time 0). Blood was collected in microvettes (Sarstedt, Montreal, Québec, Canada), and plasma separated by centrifugation at 4°C (2000g X 10 min) and stored at -80°C until assayed. Insulin was measured in plasma using a mouse ELISA kit (Linco Research, St. Charles, MO), and using mouse insulin as a standard (Crystal Chem Inc.). Insulin resistance was calculated using Homeostasis Model Assessment of insulin resistance (HOMA-IR) [25 (link)].
Microvettes
Manufactured by Sarstedt
Sourced in Germany, Sweden
Microvettes are small laboratory collection and storage tubes designed for the collection and containment of small sample volumes. These tubes are suitable for a variety of applications requiring precise and convenient handling of small-scale samples.
Automatically generated - may contain errors
Lab products found in correlation
One touch 2, by LifeScan (1 mentions)
Rat anti cd45 fitc, by Thermo Fisher Scientific (1 mentions)
Streptavidin pe cy7, by Thermo Fisher Scientific (1 mentions)
Hemavet hv950, by Drew Scientific (1 mentions)
Bio plex pro cytokine assay, by Bio-Rad (1 mentions)
Manager software 4, by Bio-Rad (1 mentions)
Radioimmunoassay kit, by MP Biomedicals (1 mentions)
Mouse rat estradiol elisa kit, by Calbiotech (1 mentions)
Infinity cholesterol, by Thermo Fisher Scientific (1 mentions)
Infinity triglyceride, by Thermo Fisher Scientific (1 mentions)
Ultra sensitive mouse insulin elisa kit, by Crystal Chem (1 mentions)
Nsg mice, by Jackson ImmunoResearch (1 mentions)
Uct ultramicrotome, by Leica (1 mentions)
Tecnai g2, by Thermo Fisher Scientific (1 mentions)
Veleta camera, by Olympus (1 mentions)
Heparin, by Sarstedt (1 mentions)
Cobas 6000, by Roche (1 mentions)
Rat mouse pinp eia, by Immunodiagnostic Systems (1 mentions)
Mouse rankl elisa kit, by Abcam (1 mentions)
Mouse osteoprotegerin elisa kit, by Abcam (1 mentions)
19 protocols using microvettes
1
Glucose Tolerance and Insulin Resistance Measurement
2
Cytokine Profiling and Thymocyte Analysis
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Peripheral blood was collected into anticoagulant EDTA-treated Microvettes (20.1341, Sarstedt) for blood collection. Cells were removed from plasma by centrifugation for 15 min at 2000× g using an ice-cold refrigerated centrifuge. Cytokines levels were measured from the resulting supernatant with LEGENDplex Mouse Inflammation Panel (13-plex) with V-bottom plates (740446, Biolegend). Analysis and quantification were performed according to the manufacturer’s protocol. Data analysis was performed using software provided by Biolegend. Manual gating was used to define beads A and B, while an automatic gating strategy was used to gate individual cytokine in APC-PE plot.
Mice were euthanized for analysis at the age of 11 weeks. To prepare single cell suspensions of thymocytes, individual thymi were placed in pre-wet 70 µm cell strainers immersed in ice-cold 2% FCS/PBS in petri dishes for gentle disruption with the end of a 5 mL syringe plunger. Thymocytes were stained with rat-anti-CD45-FITC (11–0451, eBioscience), rat-anti-CD8-Biotin (13–0081, eBioscience), and rat-anti-CD4-APC (17-0042-82, eBioscience) followed by staining with Streptavidin PE/Cy7 (SA1012, Thermo Scientific). Flow cytometry was performed using a FACSAria Fusion with FACSDiva (BD Biosciences).
Mice were euthanized for analysis at the age of 11 weeks. To prepare single cell suspensions of thymocytes, individual thymi were placed in pre-wet 70 µm cell strainers immersed in ice-cold 2% FCS/PBS in petri dishes for gentle disruption with the end of a 5 mL syringe plunger. Thymocytes were stained with rat-anti-CD45-FITC (11–0451, eBioscience), rat-anti-CD8-Biotin (13–0081, eBioscience), and rat-anti-CD4-APC (17-0042-82, eBioscience) followed by staining with Streptavidin PE/Cy7 (SA1012, Thermo Scientific). Flow cytometry was performed using a FACSAria Fusion with FACSDiva (BD Biosciences).
3
Murine Blood Collection and Cell Counting
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Blood was drawn from mice retroorbitally into EDTA-treated Microvettes (Sarstedt). Counts were generated using a Hemavet HV950 (Drew Scientific).
4
Measuring Corticosterone Levels in Rats
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Rats were injected with NMU (1 µg, ICV) or an equal volume of vehicle solution (Ringer) (n = 8 per treatment group). Twenty minutes later, capillary blood from the tail was collected in microvettes (Sarstedt, Helsingborg, Sweden). The blood was centrifuged (5 minutes, 10 000 g), and corticosterone was thereafter measured in serum with an Enzo Corticosterone Eliza kit (AH Diagnostic, Stockholm, Sweden).
5
Blood Withdrawal in Healthy Volunteers
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Blood withdrawal (150 μL) into ethylenediaminetetraacetic acid- (EDTA-) containing microvettes (Sarstedt AG & Co., Germany) was carried out during a prophylactic examination on eight volunteers (25–35 years old, four women and four men). Informed consent was obtained from each donor. All experiments were carried out in accordance with guidelines and regulations of the Federal Research and Clinical Centre of Intensive Care Medicine and Rehabilitology, V.A. Negovsky Scientific Research Institute of General Reanimatology, Moscow, Russian Federation. All experimental protocols were approved by this Institute.
6
Corticosterone Measurement in Mice
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Mice were injected with sCT (5 μg/kg, IP) or an equal volume of vehicle solution (saline solution, IP), and 30 minutes later, capillary blood from the tail was collected in microvettes (Sarstedt, Helsingborg, Sweden). The blood was centrifuged (5 minutes, 10 000 g), and corticosterone was thereafter measured in serum with an Enzo Corticosterone enzyme‐linked immunosorbent assay (ELISA) kit (AH Diagnostic, Stockholm, Sweden).
7
Cytokine Analysis in Blood and Lung Tissue
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After final blood withdrawal 3, 9 and 24 hours post-surgery, blood and lung tissue was harvested for cytokine analysis. Blood was collected in microvettes (Sarstedt AG & Co., Nümbrecht, Germany), centrifuged at 4000 g for 10 minutes, followed by another centrifugation step at 10.000 g for 1 minutes before plasma was collected and stored at -80°C. The lung was frozen in liquid nitrogen and stored at -80°C. Then the tissue was homogenized and the cells were lysed on ice (30 minutes) and centrifuged before the supernatant was collected. The protein concentration was set at 100 μg. Both in plasma and lung homogenates concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-10, monocyte chemotactic protein (MCP)-1 and keratinocyte chemoattractant (KC) were measured by a mouse multiplex cytokine kit according to the manufacturer’s protocol (Bio-Plex Pro Cytokine Assay, Bio-Rad, Hercules, CA). The analysis was performed in duplicates. Data were automatically analysed using the standard curve of cytokine standards (Bio-Plex Manager Software 4.1). Values below the detection limit of the assay were set to zero.
8
Stress-induced Corticosterone Dynamics
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Naive wildtype C57BL/6J males were single-housed. Blood samples from each animal were taken at three different time points: basal (t = 0), post stress (t = 2.00, 2:20 or 2:40 h) and recovery (t = 5.00 h). Animals were divided into 5 groups: Control 1 (t = 2.20 h, for comparison before OF); Control 2 (t = 2.40 h, for comparison after OF); Stress 1 (post stress sample taken at t = 2.20 h); Stress 2 (post stress sample taken at t = 2.40 h, after OF); Stress 3 (post stress sample taken at t = 2 h, before the interval and OF) (see Figure 4 ). Stressed animals were restrained for 2 h in darkness, had a 20 min interval, and where indicated, were subjected to the OF. Blood, from tail nicks and after decapitation for the last time point, was collected into Microvettes (Sarstedt, Germany) left to coagulate, centrifuged and the supernatant was collected and stored at −20°C until further processing. Plasma corticosterone (CORT) concentrations were measured by a commercially available radioimmunoassay kit (MP Biomedicals, Irvine, CA, USA) according to the manufacturer's instructions.
9
Plasma and CSF Sampling for Murine Studies
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For plasma sampling (at 10 and 30 dpt), the tail vein was punctured and whole blood was sucked by capillarity in EDTA filled Microvettes (Sarstedt) (10-30 μl/mouse). Samples were kept at 4°C until centrifugation (950 g for 5 min). Plasma was then collected from supernatant and stored at −80°C for subsequent analysis.
For CSF sampling (at 10 and 30 dpt), mice were deeply anesthetized with isoflurane (4% induction, 1.5% maintenance), and CSF (3-5 μl/mouse) was obtained as part of a terminal procedure from the cisterna magna, as previously described (Liu and Duff, 2008 (link)). CSF samples were initially put on dry ice and then stored at −80°C for subsequent analysis.
For CSF sampling (at 10 and 30 dpt), mice were deeply anesthetized with isoflurane (4% induction, 1.5% maintenance), and CSF (3-5 μl/mouse) was obtained as part of a terminal procedure from the cisterna magna, as previously described (Liu and Duff, 2008 (link)). CSF samples were initially put on dry ice and then stored at −80°C for subsequent analysis.
10
Plasma Metabolite Extraction Protocol
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Tail bleed samples were collected into Microvettes (Sarstedt, Germany) at baseline and after one week prior to culling. Whole blood samples were centrifuged at 1500×g for 10 mins at 4 °C and the plasma deproteinised by adding 60% 5.8 M perchloric acid (ratio 1:10, perchloric acid:plasma). Samples were vortexed and centrifuged at 1500×g for a further 10 mins. The supernatant was stored at − 80 °C until analysis (Fig. 2 ).
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