As peripheral blood is the only tissue routinely available from human subjects, the peripheral blood hemogram was also assessed in the present work in order to provide information for possible comparative study in the future. The peripheral blood collected from the right femoral artery with a heparinized syringe in vacutainer blood collection tubes containing EDTA (Venoject II, Terumo Co., Japan) were immediately subjected to differential blood cell counts (red blood cells (RBCs), white blood cells (WBCs), and blood platelets (PLTs)) and measurement of blood hemoglobin concentration using a blood cell differential automatic analyzer (SYSMEX KX-21NV, Sysmex Corporation, Japan). The data for each experimental group were obtained from three to five mice.
Kx 21nv
Manufactured by Sysmex
Sourced in Japan
The KX-21NV is a compact and automated hematology analyzer designed for small to medium-sized laboratories. It is capable of performing complete blood count (CBC) analysis, including red blood cell, white blood cell, and platelet parameters.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex 200 system, by Bio-Rad (2 mentions)
Spotchem ez sp 4430, by Arkray (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Venoject 2, by Terumo (1 mentions)
Synergy 2 multi mode microplate reader, by Agilent Technologies (1 mentions)
Dri chem 7000, by Fujifilm (1 mentions)
Spotchem ez reagent strip kenshin 2, by Arkray (1 mentions)
Spotchem ez reagent strip kidney 3, by Arkray (1 mentions)
Cl 8000, by Shimadzu (1 mentions)
Max discovery, by PSC Biotech (1 mentions)
Hemoglobin colorimetric assay kit, by Cayman Chemical (1 mentions)
Lactate pro, by Arkray (1 mentions)
I stat, by Abbott (1 mentions)
Quantichrom iron assay kit, by BioAssay Systems (1 mentions)
Ferritin, by ALPCO (1 mentions)
Bio plex pro cytokine assay, by Bio-Rad (1 mentions)
Bio plex pro 2 wash station, by Bio-Rad (1 mentions)
Progesterone kit, by Neogen (1 mentions)
Progesterone ultra kit, by Neogen (1 mentions)
11 protocols using kx 21nv
1
Peripheral Blood Hemogram Analysis in Mice
2
Hematological Analysis of EDTA-Treated Blood
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The blood used for the hematology analysis was treated with
tripotassium ethylenediaminetetraacetate (EDTA-3 K, Nacalai Tesque,
Inc., Kyoto, Japan). The hematological examination was carried out
using an automatic analyzer (Sysmex KX-21NV, Sysmex Corporation,
Hyogo, Japan) to determine the red blood cell count (RBC), hemoglobin
concentration (HGB), hematocrit level (HCT), and mean corpuscular
volume (MCV).
tripotassium ethylenediaminetetraacetate (EDTA-3 K, Nacalai Tesque,
Inc., Kyoto, Japan). The hematological examination was carried out
using an automatic analyzer (Sysmex KX-21NV, Sysmex Corporation,
Hyogo, Japan) to determine the red blood cell count (RBC), hemoglobin
concentration (HGB), hematocrit level (HCT), and mean corpuscular
volume (MCV).
3
Blood Cell Analysis in Animals
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The blood samples obtained from 10 animals in each group at the terminal necropsy were analyzed with an automatic blood cell analyzer (KX-21NV, Sysmex, Hyogo, Japan). Differential counts of leukocytes were made by a light microscopic observation of smeared specimens stained following a routine May–Grunwald–Giemsa protocol.
4
Quantification of Cardiac Biomarkers and Blood Cells
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Levels of creatine kinase-MB isoenzyme (CK-MB) were measured by ELISA (cat. no. S032; Groundwork Biotechnology Diagnosticate, Ltd., San Diego, CA, USA), according to the manufacturer's protocol, measuring the absorbance at 450 nm using a Synergy 2 multi-mode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Lactate dehydrogenase (LDH) was measured using a commercial kit (cat. no. A125; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's protocol, using a UV-752 Spectrophotometer (Shanghai Lengguang Technology Co., Ltd., Shanghai, China). Blood samples were analyzed for the quantity of white blood cells (WBC), red blood cells (RBC) and platelets (PLT), and the concentration of hemoglobin (HGB), using an automated hematological analyzer (model no. KX-21NV; Sysmex Corporation, Kobe, Japan).
5
Blood Parameter Analysis in Mice
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Blood samples from 16-week-old mice were collected from the inferior vena cava under 4 % isoflurane anesthesia. Hemoglobin concentration (Hb), hematocrit (Ht), red blood cell count (RBC) and blood urea nitrogen (BUN) were measured using an automated counter (KX-21NV; Sysmex Corporation, Kobe, Japan) and an automated biochemical analyzer (Dri-Chem 7000; Fuji Photo Film, Tokyo, Japan) as described previously [15 (link)].
6
Bronchoalveolar Lavage Fluid Analysis
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One day after the last administration of ASOs, mice were sacrificed by exsanguination under anesthesia with 2.5% isoflurane, then BALF samples were collected by making an incision in the trachea and washing the lungs twice with 0.75 mL PBS (Thermo Fisher Scientific). BALF samples from each mouse were centrifuged at 950 g for 5 min at 4°C. The total cell counts in the cell pellet were determined using a Sysmex KX-21NV (Sysmex, Kobe, Japan), and the cell population was analyzed using Diff-Quick-stained cytospin preparations, as previously described [32 (link), 33 (link)]. The supernatant was used for neutrophil-related cytokine/chemokine measurement using the Bio-Plex 200 system (BioRad, Hercules, CA, USA), with the detection limit set at 0.
7
Automated Blood and Biochemical Analysis
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The blood cell components were measured using a KX-21NV (Sysmex, Hyogo, Japan). The plasma biochemical parameters, including GPT, GOT, γ-GTP, lipid profile, albumin, blood urea nitrogen (BUN), uric acid, and creatinine levels, were measured with an automated biochemical analyzer Spotchem EZ SP-4430 (Arkray, Kyoto, Japan), the Spotchem EZ Reagent Strip KENSHIN-2 (Arkray), and the Spotchem EZ Reagent Strip Kidney-3 (Arkray) according to the manufacturers' instructions.
8
Comprehensive Blood Analysis Protocol
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The hemoglobin concentration, hematocrit level, red blood cell count, mean cell volume, mean corpuscular hemoglobin, and mean cell hemoglobin concentration in blood samples drawn from the heart were measured using an automatic hematology analyzer (KX-21NV; Sysmex, Kobe, Japan). The partial pressures of oxygen (pO2) and carbon dioxide (pCO2) were measured using an analyzer (i-STAT; Abbott Point of Care, IL, USA). Lactic acid in the blood samples drawn from the tail was measured using a simplified analyzer (Lactate Pro; Arkray, Kyoto, Japan).
Blood samples were centrifuged at 1,500 g for 10 min at 4°C to obtain serum samples. Serum haptoglobin was measured using an ELISA Kit (Life Diagnostics, PA, USA). The serum hemoglobin level was measured using an assay kit (Hemoglobin Colorimetric Assay Kit; Cayman Chemical, MI, USA). The serum level of creatine kinase was quantified using a kit (Max Discovery; Bioo Scientific, USA). Serum iron, the unsaturated iron-binding capacity (UIBC), and lactate dehydrogenase were measured using Detaminer Fe and UIBC (Kyowa Medix, Tokyo, Japan) and LDH–L (Serotec, Sapporo, Japan) with an automatic biochemical analyzer (CL-8000; Shimadzu, Kyoto, Japan). The total iron-binding capacity (TIBC) and serum transferrin saturation were calculated as follows:
Serum transferrin saturation = serum iron/TIBC × 100
Blood samples were centrifuged at 1,500 g for 10 min at 4°C to obtain serum samples. Serum haptoglobin was measured using an ELISA Kit (Life Diagnostics, PA, USA). The serum hemoglobin level was measured using an assay kit (Hemoglobin Colorimetric Assay Kit; Cayman Chemical, MI, USA). The serum level of creatine kinase was quantified using a kit (Max Discovery; Bioo Scientific, USA). Serum iron, the unsaturated iron-binding capacity (UIBC), and lactate dehydrogenase were measured using Detaminer Fe and UIBC (Kyowa Medix, Tokyo, Japan) and LDH–L (Serotec, Sapporo, Japan) with an automatic biochemical analyzer (CL-8000; Shimadzu, Kyoto, Japan). The total iron-binding capacity (TIBC) and serum transferrin saturation were calculated as follows:
Serum transferrin saturation = serum iron/TIBC × 100
9
Cytokine and hematological profile analysis
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Hematological parameters were measured by an automated hematology analyzer KX-21NV (Sysmex Corporation, Hyogo, Japan). The levels of cytokines and albumin present in serum were determined by using commercially available ELISA kits for human IL-6, mouse erythropoietin (EPO) (R&D Systems, Minneapolis, MN, USA), mouse serum amyloid A (Life Technologies Japan, Tokyo, Japan), mouse albumin (Shibayagi, Gunma, Japan), and ferritin (ALPCO Diagnostics, Salem, NH, USA). Serum iron level was determined by QuantiChrom Iron Assay Kit (BioAssay Systems, Hayward, CA, USA). Mouse interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 were measured by Bio-Plex Pro cytokine assays according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA). The assays were performed using the Bio-Plex Pro II wash station with magnetic plate carrier, and cytokines were determined by the Bio-Plex 200 System (Bio-Rad Laboratories).
10
Progesterone Measurement Protocols in Biosamples
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The levels of plasma or serum progesterone were measured using commercially available enzyme immunoassay kits after diluting the samples twice (Progesterone Ultra Kit, #402410, standard
range: 0.1–2 ng/ml, Neogen; Lansing, MI, U.S.A.). The samples with high progesterone levels (>4 ng/ml) were reanalyzed
using an enzyme immunoassay kit with a higher standard range (Progesterone Kit, #402310, standard range: 0.4–40 ng/ml, Neogen). The low progesterone levels
in the samples obtained from F3 from November 2011 to the end of the study period were determined using a fluorescence immunoassay method with a fully-automated microfluidic immunoassay
system (Mini Vidas SV-5010, SYSMEX bioMerieux; Tokyo, Japan), because the additional Progesterone Ultra Kits were not available in Japan at the time of our study. All procedures were
performed according to the manufacturer’s instructions. The intra- and inter-assay coefficients of variation were less than 10% for all three methods. The WBC counts were analyzed with an
automated hematology analyzer (KX-21NV, Sysmex; Kobe, Japan).
range: 0.1–2 ng/ml, Neogen; Lansing, MI, U.S.A.). The samples with high progesterone levels (>4 ng/ml) were reanalyzed
using an enzyme immunoassay kit with a higher standard range (Progesterone Kit, #402310, standard range: 0.4–40 ng/ml, Neogen). The low progesterone levels
in the samples obtained from F3 from November 2011 to the end of the study period were determined using a fluorescence immunoassay method with a fully-automated microfluidic immunoassay
system (Mini Vidas SV-5010, SYSMEX bioMerieux; Tokyo, Japan), because the additional Progesterone Ultra Kits were not available in Japan at the time of our study. All procedures were
performed according to the manufacturer’s instructions. The intra- and inter-assay coefficients of variation were less than 10% for all three methods. The WBC counts were analyzed with an
automated hematology analyzer (KX-21NV, Sysmex; Kobe, Japan).
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