Hema 3

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada

The Hema 3 is a fully automated hematology analyzer designed for routine clinical laboratory testing. It provides rapid and accurate analysis of complete blood count (CBC) parameters, including red blood cells, white blood cells, and platelets. The Hema 3 utilizes advanced technology to deliver reliable results, enabling efficient patient diagnosis and treatment monitoring.

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Hema 3 staining kit (39 citations) Hema 3 Manual Staining System (12 citations) Hema-3 staining system (10 citations) Hema 3 Fixative and Solutions (7 citations) HEMA 3 solution I (3 citations) Hema 3 Manual Staining Stat Pack (3 citations) Protocol HEMA-3 cell staining kit (3 citations) Protocol Hema 3 Wright-Giemsa stain (3 citations) Hema-3 reagents (2 citations) Hema-3 stain solutions (2 citations)

99 protocols using hema 3

Allergic Airway Inflammation in Mice

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Mice were sensitized on day 0 subcutaneously at the base of the tail with either 0.5 mg/ml CFA and Ova protein or 0.5 mg/ml CFA and IgG-Ova immune complexes; the final quantity of Ova was 20 μg in both cases. On days 15, 16, and 17 mice were anesthetized with isoflurane and challenged intranasally with 20 μg Ova in 50 μl PBS. Mediastinal lymph nodes (LNs), lungs, blood, and bronchoalveolar lavage (BAL) fluid were harvested on day 19. BAL was performed as previously described [20 (link)], red blood cells were lysed and total nucleated cell counts were obtained using a hemocytometer. Cytospin slides were prepared by H&E staining with HEMA 3 (Fisher) and numbers of neutrophils, lymphocytes, DC/Macs, and eosinophils quantified. Serum samples were collected on day 19 for measurement of OVA-specific IgG1 and IgG2c antibodies by ELISA as previously described [21 (link)]. Lungs were fixed, embedded in paraffin and 5 μM sections were stained with H&E.

Ciraci C., Janczy J.R., Jain N., Haasken S., Pecli e Silva C., Benjamim C.F., Sadler J.J., Olivier A.K., Iwakura Y., Shayakhmetov D.M., Sutterwala F.S, & Cassel S.L. (2016). Immune Complexes Indirectly Suppress the Generation of Th17 Responses In Vivo. PLoS ONE, 11(3), e0151252.

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Murine Hematological Characterization

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Blood collected from heart puncture was analyzed by Dept. of Veterinary Medicine hematology core at MD Anderson Cancer Center. Blood smear slides were stained with Hema-3 fixative (Fisher Healthcare) and microscopically analyzed by a hematopathologist. Splenocytes isolated from wild type, 2MX-S and 8MX-S mouse spleens were treated with RBC lysis buffer and labelled with CD19-FITC (BD Pharmingen) and CD5-PE (eBiosciences) antibodies in binding buffer. Flow analysis was carried out by flow cytometry core at MD Anderson Cancer Center.

Pant V., Larsson C.A., Aryal N., Xiong S., You M.J., Quintas-Cardama A, & Lozano G. (2017). Tumorigenesis promotes Mdm4-S overexpression. Oncotarget, 8(16), 25837-25847.

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Quantification of Neutrophil Recruitment

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Neutrophils recruitment was measured using C57BL/6J mice (8–12 wk old) according to the protocol described by Fan et al (Fan, et al., 2013 ). All samples were administrated to murine lungs via the intranasal route as 50 μL saline solutions with recombinant human MIF at 1 μg, and the anti-murine CD74 monoclonal antibody (BD Pharmigen, San Jose, CA, cat no. 555317) at 10 μg. MIF mutants and each of the MIF-covalent inhibitor complexes were also administrated as solutions containing 1 μg of sample. Each compound was incubated with MIF at 1:1 stoichiometric ratio at 4°C for 24 hours. The complete modification of MIF by each inhibitor at 24 hours was based on the second order kinetics of each inhibitor. For in vivo antagonist assays, wild-type (WT) MIF and each mutant or MIF-inhibitor complex were administrated at 1:1 and 1:5 stoichiometric ratios (WT MIF:MIF mutant or MIF-inhibitor complex). The total number of neutrophils was calculated using the differential cell count of a minimum number of 200 cells stained with HEMA 3 (Fisher Scientific). For direct comparison of two data sets, the two tailed t-test was used. The protocol followed for the mice experiments was reviewed and approved by Yale University’s Institutional Animal Care and Use Committee (IACUC).

Pantouris G., Syed M.A., Fan C., Rajasekaran D., Cho T.Y., Rosenberg EM J.r., Bucala R., Bhandari V, & Lolis E.J. (2015). An analysis of MIF structural features that control functional activation of CD74. Chemistry & biology, 22(9), 1197-1205.

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Chemotaxis Assay using Boyden's Chamber

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Chemotaxis assays were performed in a modified Boyden’s chamber with 8-μm pore polycarbonate membrane inserts (Costar Transwell; Corning Costar, Lowell, MA, USA) as described previously. In brief, cells detached with 0.25% trypsin were seeded into the upper chamber of an insert at a density of 6 × 10⁴ in 100 μl. The lower chamber was filled with pre-warmed culture medium containing test reagents. Medium supplemented with 0.5% BSA was used as a negative control. After 24 hours, the inserts were removed from the Transwell supports. The cells that had not migrated were scraped off with cotton swap from the upper membrane, and the cells that had transmigrated to the lower side of the membrane were fixed and stained with HEMA 3 (protocol, Fisher Scientific, Pittsburgh, PA) and counted on the lower side of the membrane using an inverted microscope.

Suszynska M., Poniewierska-Baran A., Gunjal P., Ratajczak J., Marycz K., Kakar S.S., Kucia M, & Ratajczak M.Z. (2014). Expression of the erythropoietin receptor by germline-derived cells - further support for a potential developmental link between the germline and hematopoiesis. Journal of Ovarian Research, 7, 66.

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Adenovirus-Mediated Overexpression of OSM

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Wildtype and IL-13-/- mice were administered 5 × 10⁷ pfu of replication deficient AdDl70 or Ad encoding OSM (AdOSM) through the endotracheal route of administration as previously published [23 (link),38 (link)]. Mice were euthanized after 5, 7, or 14 days and bled, and alveolar lavage was performed as previously described [23 (link),38 (link)]. Alveolar lavage was centrifuged, and supernatants were stored for future analysis by ELISA. Cell pellets were resuspended, counted, and subjected to cytocentrifugation at 300 rpm for two minutes. Differential counts were determined after staining with protocol Hema3 (Fisher Scientific, Ottawa, ON, Canada). Left lungs were perfused with 10% formalin and fixed for 48 h subsequent to histological preparation and histochemical staining. Right lungs were snap frozen and stored at −80 °C for RNA extraction.

Botelho F.M., Rodrigues R., Guerette J., Wong S., Fritz D.K, & Richards C.D. (2019). Extracellular Matrix and Fibrocyte Accumulation in BALB/c Mouse Lung upon Transient Overexpression of Oncostatin M. Cells, 8(2), 126.

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Characterization of Murine Hematopoietic Colonies

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BM cells were plated 1×10⁴/well in triplicates in Methocult GF M3434 (StemCell Technologies) with DAC added directly at time of plating. Colonies were scored after 12–14 days. Samples yielding <15 colonies in the untreated condition were deemed poor quality and excluded from analysis, as was one sample from the Dnmt3a^+/m group yielding colonies in excess of 3× standard deviation. For differentiation analysis, methylcellulose was solubilized using phosphate buffered saline (PBS) and ~100–200×10³ and ~250×10³ cells were harvested for flow cytometry and cytospin preparations, respectively. All immunophenotyping antibodies were from BioLegend: cKit/CD117(PE) – Clone: 2B8; CD11b(APC) – Clone: M1/70; Ter119(APC-Cy7) – Clone: TER-119; Gr1(PE-Cy7) – Clone: RB6–8C5. DAPI exclusion was used for live/dead discrimination. Data were acquired on LSR FORTESSA (BD) and analyzed using FlowJo v10.4.1. For cytospins, cells were spun in 50μL of growth media onto SuperFrost Plus microscope slides (Fisher) using CYTOPRO centrifuge (ELITech Biomedical Systems) at 500rpm for 5 minutes and stained using HEMA 3 (FISHERbrand 122–911). Slides were scanned using BZ-X810 Keyence All-in-one microscope with a 60× NA 1.40 objective.

Casellas Roman H.L., Venugopal K., Feng Y., Shabashvili D.E., Posada L.M., Li J, & Guryanova O.A. (2020). DNMT3A alterations associated with myeloid malignancies dictate differential responses to hypomethylating agents. Leukemia research, 94, 106372.

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Isolation and Culture of Alveolar Macrophages

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MH-S (ATCC CRL-2019) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and 100 U/ml of penicillin/streptomycin (P/S, Life Technologies, Rockville, MD) antibiotics in a 37°C incubator with 5% CO₂. Mouse AM were isolated by bronchoalveolar lavage (BAL) [46 (link)]. In brief, trachea was cannulated with a 20-gauge catheter; 0.9 ml BAL buffer was instilled, flushed four times, and retrieved. A total of 3.0 ml BALF was retrieved from each mouse and cytospin slides prepared with 0.5 ml BALF were stained by HEMA-3 (Fisher, Rockford, IL) to enumerate leukocyte subtypes based on their cellular and nuclear morphological properties. After centrifugation at 2, 000 rpm, AM cells were resuspended and cultured in RPMI 1640 medium as above. MLE-12 (ATCC CRL-211) were cultured in HITES medium as above. Stable TLR2 expression HEK293 cells (ATCC CRL-157) using a pUNO-TLR2 plasmid were obtained from InvivoGen (San Diego, CA) and cultured in DMEM medium as above.

Li X., He S., Zhou X., Ye Y., Tan S., Zhang S., Li R., Yu M., Jundt M.C., Hidebrand A., Wang Y., Li G., Huang C, & Wu M. (2016). Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis. PLoS Pathogens, 12(1), e1005363.

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Quantifying Macrophage and Neutrophil Isoforms

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Cytospins of 3 × 10⁵ BAL cells were created on slides, air-dried and fixed in 100% methanol, and stored at 4°C until ready for staining. One slide was stained with Hema 3 (#123-869; Fisher Scientific) to determine the percentage of macrophages and neutrophils. Slides were stained with 1 of 2 different polyclonal rabbit antihuman iNOS antibodies for fluorescence microscopy (#AB5384 that binds at the C terminus, 1:200 dilution [Millipore], or #SC-8310, clone H-174, that binds the N terminus, 1:50 dilution [Santa Cruz]) so that each volunteer had at least 1 slide stained with each iNOS antibody (at least 2 slides total stained). The supplemental materials provide details.

Huang H.J., Isakow W., Byers D.E., Engle J.T., Griffin E.A., Kemp D., Brody S.L., Gropler R.J., Miller J.P., Chu W., Zhou D., Pierce R.A., Castro M., Mach R.H, & Chen D.L. (2014). Imaging Pulmonary Inducible Nitric Oxide Synthase Expression with PET. Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 56(1), 76-81.

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Murine Model of Allergic Airway Inflammation

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Mice were sensitized on day 0 by intraperitoneal injection with either 2 mg alum (Thermo Scientific) and 20 μg Ova or 2 mg alum and IgG-Ova (20 μg Ova). On days 15, 16, and 17 mice were intranasally challenged with 20 μg Ova in 50 μl PBS. Lymph nodes, lungs, blood, and BAL fluid were harvested on day 19. BAL was performed by delivering 1 ml cold PBS into the airway via a tracheal cannula and gently aspirating the fluid. The lavage was repeated three times. The cells were stained with trypan blue to determine viability and total nucleated cell counts were obtained using a hemocytometer. Cytospin slides were prepared and percentage of neutrophils, eosinophils, lymphocytes and DC/Macs was determined after HEMA3 staining (Fisher Scientific). Ova-specific IgG1 and IgG2c and total IgE in serum was determined by ELISA at day 19 as previously described (21 (link), 22 (link)). Lungs were fixed, embedded in paraffin and 5 μM sections were stained with H&E.

Janczy J.R., Ciraci C., Haasken S., Iwakura Y., Olivier A.K., Cassel S.L, & Sutterwala F.S. (2014). Immune complexes inhibit interleukin-1 secretion and inflammasome activation. Journal of immunology (Baltimore, Md. : 1950), 193(10), 5190-5198.

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Bronchoalveolar Lavage Cell Isolation

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BAL was performed by instilling 0.9 mL of ice-cold PBS containing 2 mM EDTA per 22 g body weight into the lungs via an intratracheal catheter and carefully aspirating the fluid. The lavage was repeated 5 times with fresh buffer and the fluid samples were pooled. Cells in the BAL fluid were counted using a hemocytometer, and differential counts were determined by examining cytospins stained with Hema3 (Fisher Scientific, Kalamazoo, MI).

Gomez J.C., Dang H., Martin J.R, & Doerschuk C.M. (2016). Nrf2 modulates host defense during S. pneumoniae pneumonia in mice. Journal of immunology (Baltimore, Md. : 1950), 197(7), 2864-2879.

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