K 4500

After PRP preparation, platelets in whole blood and PRP were counted using a hematology analyzer (K-4500; Sysmex Corp., Kobe, Japan).

Basic biochemistry measurements were performed with automatic biochemical analyzers: Cobas Integra 400 plus (Roche Diagnostics, Mannheim, Germany) and Elecsys 2010 Roche. Hs-CRP amount was determined with Roche Diagnostics test; the protein electrophoresis – with Beckman, Appraise Paragon; and blood differential test – with Sysmex SF3000 and Sysmex K4500.

Physiological conditions were comparatively studied in mice that were allowed free access to the diet and being under MDR. The assessments included evaluating changes in body mass and main organ/tissue weights, and measurements of peripheral hemogram and bone marrow cellularity (number of nucleated cells). Body weight gain was monitored weekly from onset of MDR at postnatal age 4 weeks to the end of experiment at postnatal age 13 weeks. Main organ and intra-abdominal fat weights, peripheral hemogram, and femur bone marrow cellularity were measured at the end of the experiment. For analysis of hemogram, peripheral blood was collected with a heparinized syringe in vacutainer blood collection tubes containing EDTA (Venoject II, Terumo Co., Japan), then samples were immediately subjected to a differential blood cell count and hemoglobin concentration measurement using a blood cell differential automatic analyzer (SYSMEX K-4500, Sysmex Corporation, Japan). The data for each experimental subgroup were from at least 5 mice.

One hundred milliliters of equine whole blood was collected from the jugular vein with a disposable plastic syringe containing 10% sodium citrate anticoagulant (ACD-A injection, Terumo BCT Ltd., Tokyo, Japan). The blood was
equally dispensed into 10 polypropylene tubes and then centrifuged at 400 × g for 7 min at 4°C. The plasma fraction of each tube was transferred into another polypropylene tube and then centrifuged at 2,000 ×
g for 7 min at 4°C. The supernatant was removed, and the remaining 1 ml of PRP in the bottom of each tube was resuspended and collected. The concentration of platelets in the PRP was determined
using an automated blood cell counter (Sysmex K-4500, Sysmex Corp., Kobe, Japan). The obtained PRP was stored at −30°C until administered, and a single freeze-thaw cycle was used to induce platelet activation and the release of
growth factors [22 (link), 23 (link)].
Concentrations of PDGF isoform BB (PDGF-BB) and TGF-β isoform 1 (TGF-β1) in the freeze-thawed PRP were determined using ELISA kits (Quantikine Human PDGF-BB ELISA DBB00 and Quantikine Human TGF-β1 ELISA DB100B, R&D Systems,
Minneapolis, MN, U.S.A.). These kits were designed for use in samples obtained from humans but have also been validated for use in horses [3 (link), 23 (link), 26 (link)].