For plasma hormone (glucagon and c-peptide) sampling, EDTA-coated microtubes (Cat # 16.444.100, Sarstedt, Canada) were used to collect ∼200 µL of whole blood from a saphenous vein bleed using a sterile needle (25G). All blood samples were immediately centrifuged at 12000 rpm for 5 min at 4°C to enable separation of plasma from blood cells. Plasma was then removed, and samples were aliquoted and stored at −80°C for subsequent hormone analysis. Blood glucose was measured using glucometers (ContourNext® glucose meter and test strips, Ascensia Diabetes Care, Mississauga Canada) via a tail or saphenous vein bleed using a sterile 30G needle and ∼20 μl of blood. Whole blood from the saphenous vein was also used to assess hemoglobin A1c (HbA1c) level, as an indication of overall glucose control. Commercially available enzyme-linked immunosorbent assay (ELISA) hormone assay kits were used for the determination of plasma c-peptide (Crystal Chem Cat# 90055, RRID:AB_2893130) and glucagon (Mercodia Cat# 10–1271-01, RRID:AB_2737304) levels.
Edta coated microtubes
Manufactured by Sarstedt
Sourced in Canada, Switzerland
EDTA-coated microtubes are laboratory consumables designed for the collection and storage of blood samples. The microtubes are coated with the anticoagulant EDTA, which prevents the blood from clotting during the collection and handling process. These tubes are a standard item used in clinical and research settings for various blood-based analyses and diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Infinity cholesterol liquid stable reagent, by Thermo Fisher Scientific (1 mentions)
Mouse rat insulin kit, by Mesoscale (1 mentions)
Cobas mira, by Roche (1 mentions)
Convulxin, by Santa Cruz Biotechnology (1 mentions)
Ly6g bv510, by BioLegend (1 mentions)
Hemocytometer, by Hausser Scientific (1 mentions)
5 protocols using edta coated microtubes
1
Plasma Hormones Measurement Protocol
2
Plasma Cholesterol Quantification
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Blood was collected following a 4 h fast by saphenous vein bleed or cardiac puncture at the time of sacrifice in EDTA-coated microtubes (Sarstedt). Blood was centrifuged at 900xg for 10 min at 4°C, and the plasma was collected. Total plasma cholesterol was measured using the Infinity Cholesterol Liquid Stable Reagent (Thermo Scientific) as per the manufacturer's recommendation.
3
Plasma Metabolite Measurement Protocol
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Post prandial trunk blood samples were collected in micro tubes containing 0.5 M EDTA after a 2 h fast in the dark phase; fasting blood samples (approximately 50 μL) were collected from the lateral tail vein in EDTA coated micro tubes (Sarstedt, Switzerland), after 6 h of fasting beginning at the onset of dark phase. Blood samples were centrifuged at 8700g for 10 min at 4 °C, and plasma were collected and stored at −80 °C until required. Plasma TAG, free glycerol, non-esterified fatty acids (NEFA), and β-hydroxybutyrate (BHB) concentrations were measured using standard colorimetric and enzymatic methods adapted for the Cobas MIRA auto analyzer (Hoffman LaRoche, Basel, Switzerland) [29] (link). Final TAG values were obtained by subtracting the free glycerol values from the measured TAG values. Plasma insulin levels were measured using the Mouse/Rat Insulin Kit (Meso Scale Discovery, USA #K152BZC-2) according to manufacturer's instructions.
4
Quantifying Platelet-Neutrophil Aggregation
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Whole-blood samples from seven to 10-day-old mice were obtained by cardiac puncture into EDTA coated microtubes (Sarstedt). To measure platelet–neutrophil aggregates (PNAs), we diluted whole blood 1:10 with M-199 serum-free media (12–117F; Lonza) containing 100 U/mL heparin.25 ,26 Neutrophils were labeled with Ly6G BV510 (Biolegend) and platelets were labeled with APC anti-mouse CD41 (BD Biosciences) for 15 min at 37 °C. For platelet activation studies, staining was performed in the presence of 50 ng/mL of convulxin (Santa Cruz). Samples were fixed with FACS lysis buffer, centrifuged at 500 x g for 10 min and resuspended in PBS before analysis.
5
Isolation of Murine Spleen and Bone Marrow
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Animals were anesthetized with a mixture of 400 mg/kg ketamine and 40 mg/kg xylazine. Blood was collected via cardiac puncture using a 22-gauge syringe and immediately transferred into EDTA-coated microtubes (Sarstedt). Blood samples were then put on slow rotation at room temperature until processing. Prior to collection of the spleen and femurs, animals were transcardially perfused with cold Hanks’ balanced salt solution (HBSS) to remove blood from the vasculature.
Spleens were harvested from anesthetized animals and placed in HBSS (without Ca2+/Mg2+). Spleens were homogenized and passed through a 70-µm nylon mesh strainer (BD Biosciences). Erythrocyte lysis was performed using the ACK buffer. The cell suspension was passed on a second 70-µm nylon mesh strainer and the cell count measured.
For the bone marrow, animals were anesthetized and their left femurs isolated and flushed with HBSS (without Ca2+/Mg2+) + 2% FBS using a 25-gauge needle. Erythrocytes were lysed using the ACK buffer (NH4Cl 150 mM, potassium bicarbonate 10 mM, EDTA 0.01 mM). Cells were manually counted with a hemocytometer (Hausser Scientific).
Spleens were harvested from anesthetized animals and placed in HBSS (without Ca2+/Mg2+). Spleens were homogenized and passed through a 70-µm nylon mesh strainer (BD Biosciences). Erythrocyte lysis was performed using the ACK buffer. The cell suspension was passed on a second 70-µm nylon mesh strainer and the cell count measured.
For the bone marrow, animals were anesthetized and their left femurs isolated and flushed with HBSS (without Ca2+/Mg2+) + 2% FBS using a 25-gauge needle. Erythrocytes were lysed using the ACK buffer (NH4Cl 150 mM, potassium bicarbonate 10 mM, EDTA 0.01 mM). Cells were manually counted with a hemocytometer (Hausser Scientific).
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