The mice were administered 0.5 mg/kg of peptide via IP injection. Four h after injection, blood was acquired by venesection into lithium heparin-coated microvette tubes (Sarstedt, Germany). In a separate study, male Sprague Dawley rats (average weight 250 g, obtained from Charles River) were administered 4 mg/kg of peptide SC mixed in aqueous ZnCl2 solution to a molar ratio of 0.7:1 (peptide:ZnCl2), and blood was collected by venesection at several time points up to 72 h. Plasma was separated by centrifugation at 10,000 g for 8 min at 4 °C. Plasma concentrations were assessed by radioimmunoassay using an in-house assay as previously described [35 (link)] using standard curves generated from each SRB103 peptide to ensure that equivalent recovery was obtained.
Lithium heparin coated microvette tubes
Manufactured by Sarstedt
Sourced in Germany
Lithium heparin-coated microvette tubes are laboratory equipment designed for the collection and storage of blood samples. The tubes are coated with lithium heparin, an anticoagulant that prevents blood from clotting during the collection and handling process.
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8 protocols using lithium heparin coated microvette tubes
1
Pharmacokinetic Evaluation of SRB103 Peptide
2
Plasma Parameter Measurements in Mice
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For measurements of the blood plasma parameters 10 wild type and 11 HCN3−/− animals were used. The mice were restrained and blood samples were collected from the lateral tail vein into lithium heparin-coated Microvette tubes (Sarstedt). Blood samples were centrifuged for 10 min at 3,000 rpm and 4°C, and 150 μl of plasma were stored on −20°C until investigation. Plasma parameters were analyzed with a Hitachi 717 autoanalyzer and reagents from Roche after diluting the plasma samples 1:2 with H2O as previously described (Rathkolb et al., 2000 (link)).
3
Blood Collection and Analysis
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PB from mice was retro-orbitally collected into lithium heparin-coated Microvette tubes (Sarstedt), and the tubes were gently rotated for 10 min. Complete blood cell counts were analyzed using a Hemavet 950FS (Drew Scientific) according to the manufacturer’s instructions.
4
Submandibular Blood Collection in Mice
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Mice anesthetised with isoflurane were punctured for submandibular blood collection with a 5 mm lancet (Medipoint, Inc., Mineola, NY) and blood was collected in Lithium heparin-coated microvette tubes (Sarstedt, Numbrecht, Germany). Samples were centrifuged to determine the packed cell volume and quantified in a blinded manner by the Vanderbilt University Medical Center Comparative Pathology Laboratory.
5
Measuring Plasma Catecholamines Pre and Post Exercise
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Approximately 500–600 μL of capillary blood was collected Pre and Post exercise through a finger prick. A 17-gauge 2.0 mm depth disposable lancet was used to induce bleeding on either the third or fourth finger. The first drops of blood were used to measure blood lactate using a lactate meter (Lactate Plus Meter, Nova Biomedical, Waltham, MA, USA). Then, a massage technique was used to promote bleeding and the remaining volume of blood was collected via capillary action into lithium-heparin coated microvette® tubes (SARSTEDT, Newton, NC, USA). Whole blood was then centrifuged at 10,000 rpm for 10 min, plasma was decanted, and subsequently stored at −80° C until biochemical analysis which was completed following the conclusion data acquisition. Plasma concentrations of EPI and NE were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (ABNOVA, Taipei, Taiwan) [15 (link),16 (link)]. All samples were analyzed in duplicate and according to the manufacturer’s instructions.
6
Glucose Tolerance Test with Peptide
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The mice were fasted for at least 4 h before commencing the glucose tolerance test depending on the peptide treatment length. Peptide or vehicle (0.9% saline) was injected into each mouse's intraperitoneal (IP) cavity either 8 h before, 4 h before, or at the same time as the glucose challenge (acute). Glucose was dosed at 2 g/kg of body weight. Blood glucose levels were measured before a glucose challenge then at the times as indicated in the figure using a hand-held glucose meter (GlucoRx Nexus). Blood samples for insulin were collected at 10 min into lithium heparin-coated microvette tubes (Sarstedt, Germany), followed by centrifugation (10,000 RPM for 8 min at 4 °C) to separate the plasma. Plasma insulin was measured using a Cisbio mouse insulin HTRF kit.
7
Glucose tolerance test with peptide
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Mice were fasted for at least 4 hours before commencing the glucose tolerance test, depending on the peptide treatment length. Mice were injected into the intraperitoneal (IP) cavity with peptide or vehicle (0.9% saline) either 8 hours before, 4 hours before, or at the same as the glucose challenge (acute). Glucose was dosed at 2 g/kg body weight. Blood glucose levels were measured before glucose challenge, then at the times as indicated in the figure using a hand-held glucose meter (GlucoRx® Nexus). Blood samples for insulin were collected at 10 minutes into lithium heparincoated microvette tubes (Sarstedt, Germany), followed by centrifugation (10,000 RPM, 8 min, 4°C) to separate plasma. Plasma insulin was measured using the Cisbio mouse insulin HTRF kit.
8
Plasma Peptide Quantification in Mice
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Mice were administered 0.5 mg/kg peptide via IP injection. 4 hours after injection, blood was acquired by venesection into lithium heparin-coated microvette tubes (Sarstedt, Germany). Plasma was separated by centrifugation at 10,000 g for 8 minutes at 4°C. Plasma concentrations were assessed by radioimmunoassay using an in-house assay as previously described [33] (link), using standard curves generated from each SRB103 peptides to ensure equivalent recovery was obtained.
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