Mice were anesthetized by isoflurane inhalation and then bled retroorbitally using heparinized micro-hematocrit capillary tubes (Fisherbrand). Twenty to fifty μl of blood were collected in 1 ml eppendorff tubes and centrifuged for 10 min at 10,000 rpm to separate the serum. Serum immunoglobulin titers were measured by ELISA. Polystyrene microtiter plates (Nunc) were coated overnight with 50 μg/ml of ovalbumin (OVA) protein (Sigma), then washed and blocked with bovine serum albumin (1%). Serum samples were applied at 1:5 dilution, and incubated for 3 hours at room temperature, then washed and incubated with alkaline-phosphatase-goat-anti-mouse IgM, IgA, IgG1 (1:100) developed by the addition of p-nitrophenyl phosphate solution (Sigma-Aldrich). Optical density (OD) 490 nm was measured using a Molecular Devices spectrophotometer.
Microhematocrit capillary tube
Manufactured by Thermo Fisher Scientific
Sourced in United States
Microhematocrit capillary tubes are hollow, glass tubes used to collect and measure the volume percentage of red blood cells in whole blood samples. These tubes are designed for quick and accurate determination of hematocrit levels.
Automatically generated - may contain errors
Lab products found in correlation
Polystyrene microtiter plate, by Thermo Fisher Scientific (2 mentions)
Ovalbumin ova protein, by Merck Group (2 mentions)
P nitrophenyl phosphate solution, by Merck Group (2 mentions)
Isosol, by Vedco (2 mentions)
2 acryloyloxyethyl isocyanate, by Resonac (2 mentions)
Ethanol l3k kit, by Sekisui (2 mentions)
Ultra sensitive mouse insulin elisa kit, by Crystal Chem (2 mentions)
Hemavet 950fs, by Drew Scientific (2 mentions)
Wako diagnostics kits for cholesterol e, by Fujifilm (2 mentions)
Procyte dx hematology analyzer, by IDEXX (2 mentions)
Procyte dx, by IDEXX (2 mentions)
Heparin, by Thermo Fisher Scientific (2 mentions)
Istat cg4 cartridge, by Abbott (2 mentions)
I stat 1 handheld analyzer, by Abbott (2 mentions)
Corticosterone eia kit, by Enzo Life Sciences (2 mentions)
Z gel microtube, by Sarstedt (2 mentions)
C57bl 6ncrl, by Jackson ImmunoResearch (1 mentions)
C57bl 6j, by Jackson ImmunoResearch (1 mentions)
Model p 87, by Sutter Instruments (1 mentions)
Accu chek active, by Roche (1 mentions)
75 protocols using microhematocrit capillary tube
1
Serum Immunoglobulin Quantification by ELISA
2
Blood Glucose Measurement in Mice
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Blood was collected from mice aged 4–8 months old via submandibular bleeding using heparinized microhematocrit capillary tubes (Fisher #22-362-566) (n = 5 for each group of mice). Blood collected was applied onto the test strip of the Accu-chek Guide Meter. Statistical analysis performed using Student’s t test.
3
Blood Collection and Hematological Analysis
4
Lipoprotein(a) Clearance in Sort1 Knockout Mice
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Mice bearing a Sort1 null allele (48 (link)) on a C57BL/6NCrl background were sourced from Valerie Wallace (Ottawa Hospital Research Institute) and were backcrossed onto C57BL/6J (Jackson Laboratories) over eight generations. Mice were housed in a 12 h-light/dark cycle and fed chow diet (2018 Teklad Global; Harlan Laboratories). The IRCM animal care committee approved all procedures. Heterozygous animals were intercrossed to yield Sort1+/+ and Sort1-/- littermates that were used in clearance experiments. After 3 h fasting with a free access to water, mice were injected in the tail vein with 25 μg of human Lp(a) in 100 μl of saline. Each mouse was bled after injection at the tip of the tail at 5, 15, 30, 120, and 360 min into heparin-coated capillaries (22-362-566, Microhematocrit Capillary Tubes, Fisher Scientific). For Lp(a) injections, food was returned after 120 min. Blood was transferred to Eppendorf tubes on ice and centrifuged at 3800 g for 10 min at 4°C. Plasma was assessed immediately or stored at −80°C. Plasma Lp(a) concentrations were determined by an Lp(a) ELISA kit (Mercodia).
5
Whole-cell Patch-clamp of Primate Cortical Neurons
6
Glucose and Triglyceride Metabolism
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At both 2 and 6 months of age, female IL-15tg, B6, and IL-15−/− mice were administered 2 g/kg D-glucose or 10 µl/g olive oil following an overnight fast. The olive oil gavage was performed in either the absence or presence of the lipase inhibitor tyoxapol (Sigma, St. Louis, MO, USA), where mice were administered 0.5 mg/g intravenously immediately after gavage. Blood samples were collected by submandibular bleeds at baseline (time 0) and at various time points post-gavage from each mouse per group. Blood glucose was measured using a hand-held glucometer (Accu-Chek Active, Roche Diagnostics, Laval, Canada). Following the olive oil gavage, blood was passed through heparinized micro-hematocrit capillary tubes (Fisher Scientific, Pittsburgh, PA, USA), collected in eppendorf tubes, and centrifuged at 12,000 rpm for 10 minutes at 4°C to collect plasma. Triglyceride plasma levels were measured using the L-type triglyceride M colorimetric assay (Wako Diagnostics, Richmond, VA, USA). The area under the curve for the total glucose and triglyceride responses was assessed using the trapezoidal rule. Linear regression analysis was used to calculate slopes for triglyceride responses in tyoxapol treated mice.
7
Oral Glucose Tolerance Test in Pigs
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Two weeks prior to OGTT, pigs were introduced to a standard meal consisting of 50 g ground pregrower fodder, and acclimated to handling and standing in a sling. OGTT was conducted after overnight fasting. An hour before OGTT, ears were cleaned and Lidocaine/prilocaine cream applied (EMLA Cream; AstraZeneca, Mississauga, Ontario) to reduce pain. After measuring fasting blood glucose (FBG) animals were given the standard meal (50 g) mixed with 2 g/kg glucose solution. Upon finishing the meal (time 0), glucose was measured with a glucometer (Accu-Check Compact Plus; Roche Diagnostics) in whole blood from ear vein at 15, 30, 45, 60, 90, 120, and 180 min. Additional blood samples were collected - during the first 45 min by 70 μl microhematocrit capillary tubes coated with ammonium heparin (Fisher Scientific). Blood samples were centrifuged at 1500 rpm for 10 min at 4 °C and plasma collected and stored at −80 °C until assayed for insulin by ELISA, according to the manufacturer’s instructions (Alpco Diagnostics, Salem, N.H., USA). Blood glucose and plasma insulin concentrations were plotted as a function of time, and area under the curve was calculated using established methods72 (link).
8
Measuring Serum CTX-I and EdU Labeling in Mice
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For serum CTX-I assays, serum was collected through retro-orbital bleeding from mice starved for 6 h, and analyzed with the RatLaps ELISA kit (Immunodiagnostic Systems, Ltd., Gaithersburg, MD, USA) according to manufacturer's instructions. To collect serum, blood was collected with heparinized micro-hematocrit capillary tubes (22–362–566, Fisher Scientific, Pittsburgh, PA, USA), transferred to BD Microtainer SST Tubes (365967, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The SST tubes containing blood samples were then inverted five times and let sit at room temperature for 30 min to allow clotting before centrifugation for 90 s.
EdU (Invitrogen, Carlsbad, CA, USA) dissolved in water was injected intraperitoneally at 10 μg·g−1 body weight at 4 h before collection. Frozen sections were subjected to immunostaining for Osx (ab22552, Abcam, Cambridge, MA, USA) and Alexa Fluor 647 conjugated goat anti-rabbit secondary antibody (A21246, Invitrogen), followed by a click reaction according to manufacturer’s instructions (Click-iT EdU Alexa Fluor 488 Imaging Kit, Invitrogen). A non-immune IgG (5415S, Cell Signaling Technology, Danvers, MA, USA) was used as negative control. Images were acquired with the Nikon C-1 confocal system.
EdU (Invitrogen, Carlsbad, CA, USA) dissolved in water was injected intraperitoneally at 10 μg·g−1 body weight at 4 h before collection. Frozen sections were subjected to immunostaining for Osx (ab22552, Abcam, Cambridge, MA, USA) and Alexa Fluor 647 conjugated goat anti-rabbit secondary antibody (A21246, Invitrogen), followed by a click reaction according to manufacturer’s instructions (Click-iT EdU Alexa Fluor 488 Imaging Kit, Invitrogen). A non-immune IgG (5415S, Cell Signaling Technology, Danvers, MA, USA) was used as negative control. Images were acquired with the Nikon C-1 confocal system.
9
Cytokine and Autoantibody Profiling in Mice
10
Blood Serum Extraction for Ghrelin Analysis
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Serum samples were obtained from live mice by collecting blood from the tail vein. Mice were warmed via a heat lamp, and a small nick was made in the dorsal portion of the tail vein. Heparinised microhematocrit capillary tubes (Fisherbrand™) were used for blood collection. Blood samples were centrifuged at 3000g for 5 min in a tabletop centrifuge. Serum was removed via micropipette and was stored at −80°C for future analyses. For serum ghrelin measurements, mice were fasted overnight (12 h) before the first collection and were subsequently allowed to feed ad libitum (4 h) before the second collection.
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