Folllowing white blood cell count (WBC) measurement on a Coulter®Ac.T diff analyzer (Beckman Coulter, Fullerton, CA, USA), flowcytometry was performed on whole blood [after red blood cell lysis with ammonium chloride] using an LSR Fortessa™ (BD Biosciences, San Jose, CA). Absolute cell counts of monocytes, natural killer (NK) cells, T cells, and B cells were calculated from WBC numbers using Infinicyt software (Cytognos, Salamanca, Spain). Lymphocytes were first gated based on FSC / SSC characteristics, and B cells were defined by expression of the pan-B cell marker CD19. Further gating was performed for defined CD19+ B cell subpopulations, i.e. transitional B cells (CD38hi/CD27-), naive mature B cells (CD38−/CD27−/IgM+/IgD+), non-switched memory B cells (CD38−/CD27+/IgM+/IgD+), IgM-only B cells (CD38−/CD27+/IgM+/IgD-), switched memory B cells (CD38−/CD27+ or −/IgM−/IgD-IgG+ or IgA+ or IgE+), plasma blasts (CD38hi/CD27+), CD5+/CD43+ B cells (CD38−/CD5+/CD43+), and CD21low B cells (CD38dim/CD21low) according to published data [28 (link)] (see also (Additional file 6 : Table S1), using 11-color flowcytometric stainings (Additional file 6 : Table S3) and FACS DIVA software (BD Biosciences) for analysis.
Coulter act diff analyzer
Manufactured by Beckman Coulter
Sourced in United States, Germany, Australia
The Coulter AcT diff Analyzer is a compact hematology analyzer designed to perform complete blood count (CBC) and 3-part differential analysis. It provides accurate and reliable results for a range of routine clinical laboratory tests.
Automatically generated - may contain errors
Lab products found in correlation
Attune flow cytometer, by Thermo Fisher Scientific (2 mentions)
Eosin 5 maleimide, by Thermo Fisher Scientific (2 mentions)
Facsdiva software, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Infinicyt software, by Cytognos (1 mentions)
Facscanto 10c, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by FlowJo (1 mentions)
Thrombin reagent, by Siemens (1 mentions)
Quantikine high sensitivity elisa, by R&D Systems (1 mentions)
Cobas 8000 modular analyzer, by Roche (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Easysep mouse mesenchymal stem progenitor cell enrichment kit, by STEMCELL (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
I stat, by Abbott (1 mentions)
19 protocols using coulter act diff analyzer
1
Comprehensive B cell phenotyping by flow cytometry
2
Automated Complete Blood Count Analysis
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Automated CBC analysis was performed on the fraction of retained blood using the COULTER Ac·T diff Analyzer (Beckman). All samples were run in duplicate. Alternatively, a spreader slide and 10 μL of blood were used to create a peripheral blood smear. Air-dried slides were then stained with Wright’s Giemsa using an immersion protocol. Briefly, slides were stained for 1 minute, rinsed for 5 minutes in phosphate buffer (pH 6.8) (made with potassium phosphate, monobasic 50.1% [w/w] and sodium phosphate, dibasic 49.9% [w/w]), washed briefly in running deionized water, dried, and coverslipped. The WBC count was determined by taking the average number of WBCs in 10 fields at ×40 high power and multiplying by 2.0 × 109/L (67 (link)).
3
Tumor Dissociation and Blood Analysis
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Implanted tumors were mechanically dissociated into suspensions of individual spleen or tumor cells expressing tissue fragments through a 70-μm cellular sieve with a plunger. Cell suspensions were centrifuged, counted, and washed once with PBS. Blood samples were drawn from the tail vein according to IACUC Guidelines. Peripheral blood was counted using a Coulter AcT diff Analyzer (Beckman, Miami, FL).
4
Neutrophil Migration Measurement in SIRS and Sepsis
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Neutrophil migration was determined as previously described (Alves-Filho et al., 2010 (link)). Peritoneal cavity lavage (PCL) was performed 6 h after LPS-induced SIRS or CLP-induced sepsis using 3 ml of phosphate buffer saline/ethylene diamine tetraacetic acid (PBS/EDTA) 1 mM and peritoneal fluids were collected. A Coulter AcT Diff analyzer (Beckman Coulter) was used to perform total cells counts. Differential cell counts were determined on Cytospin slides stained with Panótico Rápido LB dye (Laborclin, Brazil). The results are expressed as the mean number of neutrophils ± SEM per cavity.
5
Nitric Oxide Exposure Effects on Mouse Blood
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Wild-type mice (C57BL/6, body weight, >25 g) were housed in a viral-free environment in standard approved chambers (5 mice/cage). Mice were allowed to breathe 8 parts per million (ppm) NO gas for 8 weeks as described [10 (link)]. To determine expression levels of the transgenes or investigate the effects of cGMP signaling on mouse blood cells, mice were anesthetized with ketamine/xylazine (0.1mg/0.015mg/g, IP) and euthanasia was performed by cervical dislocation with anesthesia. Blood was obtained from tail veins and bone marrow (BM) cells were harvested from femur bones. cGMP levels of red blood cells (RBCs) were measured using an ELISA kit (Cayman Laboratories, Ann Arbor, MI, USA) as described [28 (link)]. Complete blood count was performed using an automatic blood cell analyzer (Coulter Ac•T diff analyzer, Beckman Coulter, Fullerton, CA, USA). All animal studies were approved by the Institutional Animal Care and Use Committees of the Georgia Regents University and the University of California San Francisco.
6
Evaluating SM Cytotoxicity on RH30 and NK Cells
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To determine at which concentrations SM become toxic to RH30 and NK cells, the cells were challenged with increasing doses of SM, harvested, and stained with 4′,6-diamidino-2-phenylindole (DAPI; BioLegend, USA) after 24 and 48 h. The cell suspensions were measured by flow cytometry using a BD FACSCanto10c™ instrument (BD Bioscience, San Diego, CA, USA) and data were analyzed using FlowJo (FlowJo LLC, Ashland, Oregon, USA), first gating on single cells, then defining the DAPI negative population as viable cells. To analyze the effect of SM on the proliferation of NK cells, the cells were isolated and taken into culture as described above with increasing doses of SM in addition to IL-2. On days 0, 3, 6, and 10, NK cells were harvested and counted using the COULTER® Ac·T diff™ Analyzer (Beckman Coulter, Germany), an automatic cell counter.
7
Anesthetized Blood Collection and Analysis
8
Evaluating Clot Analogue Mechanics
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Donor age and sex was recorded. RBCs, platelets and white blood cells were counted with a Coulter Counter (COULTER® AC•T diff™ Analyzer, Beckman Coulter, CA) (Fig. 1 e). Fibrinogen levels were measured using the Clauss assay (Thrombin Reagent, Siemens Healthineers, Erlangen, Germany) on the Sysmex CS5100 coagulation analyzer (Siemens Healthineers, Diagnostics B.V., Newark, DE, USA) (Fig. 1 f). Since contraction has been shown to alter clot analogue mechanics, the degree of clot contraction was assessed gravimetrically as the percentage weight of the expelled serum compared with the weight of the total blood mixture (Fig. 1 g).13 (link)
9
Biomarkers and Inflammatory Cytometry
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C‐reactive protein (CRP) and other soluble and cellular biomarkers were measured in blood as part of the baseline collection in the CLSA. C‐reactive protein and albumin were measured in serum using the Cobas 8000 modular analyzer (Roche Diagnostics), TNF and IL‐6 were measured in serum using the Quantikine high sensitivity ELISA (R & D Systems), and absolute measures of monocytes and granulocytes were obtained in whole blood using the Coulter Ac·T diff Analyzer (Beckman Coulter). All measures were log‐transformed and standardized as above.
10
Isolation and Characterization of Murine Bone Marrow and Splenic Cells
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BM was flushed from femurs using phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS). Spleens were harvested from mice and processed in PBS and 2% FBS using the Miltenyi gentleMACS and a C-type tube (Bergisch Gladbach, Germany). Blood was collected via cardiac puncture into 3.2% sodium citrate. Each fraction was counted using a Coulter AcT Diff Analyzer (Beckman Coulter).
To isolate BM stromal/endothelial cells, bones were harvested from NSG and C57BL/6 mice. BM was flushed and discarded and the bones were treated with 1 mg/mL collagenase type-1 (Worthington) as previously described.23 (link) Blood cells were depleted using the EasySep™ Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (Cat. n. 19771 StemCell Technologies) following the manufacturer’s protocol.
BM-MSC were isolated from NSG femurs using a modification of a previously described protocol24 (link) (see Online Supplementary Methods).
To isolate BM stromal/endothelial cells, bones were harvested from NSG and C57BL/6 mice. BM was flushed and discarded and the bones were treated with 1 mg/mL collagenase type-1 (Worthington) as previously described.23 (link) Blood cells were depleted using the EasySep™ Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (Cat. n. 19771 StemCell Technologies) following the manufacturer’s protocol.
BM-MSC were isolated from NSG femurs using a modification of a previously described protocol24 (link) (see Online Supplementary Methods).
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