Hema3 kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Hema3 kit is a laboratory equipment product designed for automated hematology analysis. It provides accurate and reliable results for red blood cell, white blood cell, and platelet count measurements, as well as hemoglobin determination.

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Lab products found in correlation

39 protocols using hema3 kit

Lung Immune Cell Profiling

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Following euthanasia, mouse lungs were lavaged with 1 mL cold PBS and the recovered fluid was collected and immediately placed on ice. BALF volumes were recorded and the total number of cells quantified by counting on a haemocytometor. The volume of BALF required for 25,000 cells total per sample was spun onto glass slides using a cytospin (ThermoFisher). All slides were stained using a HEMA III kit (ThermoFisher) and 200 cells per slide were counted and identified as monocytes, lymphocytes, neutrophils, eosinophils, or basophils. All samples were counted duplicate and the average values were calculated.

Chen H., Zheng X., Nicholas J., Humes S.T., Loeb J.C., Robinson S.E., Bisesi JH J.r., Das D., Saleh N.B., Castleman W.L., Lednicky J.A, & Sabo-Attwood T. (2017). Single-walled carbon nanotubes modulate pulmonary immune responses and increase pandemic influenza a virus titers in mice. Virology Journal, 14, 242.

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Differential Cell Count in Mouse BALF

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After euthanasia, mouse lungs were lavaged with 1 mL ice-cold PBS and the recovered fluid was immediately placed on ice. The volume and cell count of each BALF sample was recorded. The volume of BALF required for 25,000 total cells per sample was calculated and spun onto glass slides using Cytospin (ThermoFisher, Waltham, MA). All slides were stained with HEMA III kit (ThermoFisher, Waltham, MA) and 200 cells on each slide were counted and identified as macrophages, neutrophils, eosinophils, basophils, and lymphocytes using light microscopy. All slides were blinded and counted by two individuals and the average values were calculated.

Chen H., Humes S.T., Rose M., Robinson S.E., Loeb J.C., Sabaraya I.V., Smith L.C., Saleh N.B., Castleman W.L., Lednicky J.A, & Sabo-Attwood T. (2020). Hydroxyl functionalized multi-walled carbon nanotubes modulate immune responses without increasing 2009 pandemic influenza A/H1N1 virus titers in infected mice. Toxicology and applied pharmacology, 404, 115167.

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Mouse Lung Lavage and Cell Analysis

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Immediately following euthanasia, mouse tracheas were cannulated and the lungs lavaged three times with 0.5 ml of PBS containing 0.6 mM EDTA. Cells were counted using a hemacytometer and spun on to slides using a Cytospin 4 (Thermo Fisher Scientific, Waltham, MA). The slides were stained with the Hema 3 kit (Thermo Fisher) and used for differential counts of macrophages, lymphocytes, and neutrophils. Remaining lavage fluid was divided into two aliquots. One portion was centrifuged at 300×g for 10 min at 4 °C for subsequent studies of cytokines and other soluble factors, and the other portion was stored without centrifugation to preserve high molecular weight mucins that sediment at low centrifugation speeds. Both were frozen on dry ice and stored at −80 °C.

Hancock L.A., Hennessy C.E., Solomon G.M., Dobrinskikh E., Estrella A., Hara N., Hill D.B., Kissner W.J., Markovetz M.R., Grove Villalon D.E., Voss M.E., Tearney G.J., Carroll K.S., Shi Y., Schwarz M.I., Thelin W.R., Rowe S.M., Yang I.V., Evans C.M, & Schwartz D.A. (2018). Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice. Nature Communications, 9, 5363.

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BALF Cell Differential Analysis

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BALF were briefly centrifuged at 150 x g and the supernatant was collected for analysis. Pelleted cells were resuspended in PBS and cells were counted using a hemocytometer prior to loading into an EZ Cytofunnel (Thermo Scientific) and centrifuged at 600 RPM for 10 minutes. Slides were fixed and stained using the Hema 3 kit (Thermo Scientific) and cell differentials were counted based on at least 200 cells per slide.

Dustin C.M., Habibovic A., Hristova M., Schiffers C., Morris C.R., Lin M.C., Bauer R.A., Heppner D.E., Daphtary N., Aliyeva M, & van der Vliet A. (2021). Redox-dependent activation of Src kinase mediates epithelial IL-33 production and signaling during acute airway allergen challenge. Journal of immunology (Baltimore, Md. : 1950), 206(12), 2989-2999.

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Analysis of BAL Cytokines and Mucins

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BAL fluids were harvested by 3 successive instillations of 500 μL of PBS into the lungs, and pooled BAL fluids were briefly centrifuged at 150 × g and supernatants was collected for analysis of cytokines and other secreted mediators. BAL levels of IL-33, IL-5, IL-13, IL-17A, IL-1β and amphiregulin (AREG) were assessed using DuoSet ELISA Kits (R&D Systems; DY3626, DY405, DY413, DY1399, DY401, DY989) according to manufacturer protocol and were read on a BioTek Synergy HT plate reader. MUC5AC in BAL was analyzed using a Mouse Mucin-5 Subtype AC ELISA kit (MyBioSource; MBS265057), according to the manufacturer’s instructions. Pelleted BAL cells were resuspended in PBS and counted using a hemocytometer prior to loading into an EZ Cytofunnel (Thermo Scientific) and centrifuged at 600 RPM for 10 minutes. Cytospin slides were fixed and stained using the Hema 3 kit (Thermo Scientific) and cell differentials were counted based on at least 200 cells per slide.

Morris C.R., Habibovic A., Dustin C.M., Schiffers C., Lin M.C., Ather J.L., Janssen-Heininger Y.M., Poynter M.E., Utermohlen O., Krönke M, & van der Vliet A. (2022). Macrophage-intrinsic DUOX1 contributes to type 2 inflammation and mucus metaplasia during allergic airway disease. Mucosal immunology, 15(5), 977-989.

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Bronchoalveolar Cell Isolation and Analysis

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Mouse bronchoalveolar cells were isolated by lavage of the lungs with 1 ml PBS. Cells were then counted, spun onto slides, and stained with Hema-3 kit (Thermo Fisher). Differential cell counts were conducted in a blinded manner using standard morphological criteria.

Saravia J., You D., Shrestha B., Jaligama S., Siefker D., Lee G.I., Harding J.N., Jones T.L., Rovnaghi C., Bagga B., DeVincenzo J.P, & Cormier S.A. (2015). Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33. PLoS Pathogens, 11(10), e1005217.

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Transwell Invasion Assay for MPC1 and MPC2

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A375 or SK-MEL-28 cells were transfected with scramble or empty vector, or PCDNA3 vector containing MPC1 and MPC2, or siRNAs against to MPC1 and MPC2 for 48 h. Then, the transfected 1×10⁵ cells seeded on the upper side of a Matrigel-coated 6.5 mm polycarbonic Transwell chamber (Corning Costar, USA) with serum-free DMEM culture medium, and the lower side of the Transwell chamber was filled with DMEM culture medium with 10% FBS. For the 2-DG treatment group, 10 mM 2-DG was added to the upper side. After incubation for 24 h, the cells that stayed on the upper surface of the chamber were removed with cotton swabs. We washed the chamber with 1XPBS 2 times and stained the penetrated cells on the lower surface of the chamber using a Hema 3 kit (Thermo Scientific, Rockford, IL, USA) following the manufacturer’s instruction. Images were taken with a NikonTS-100 microscope.

Deng Y., Li H., Yin X., Liu H., Liu J., Guo D, & Shi Z. (2018). C-Terminal Binding Protein 1 Modulates Cellular Redox via Feedback Regulation of MPC1 and MPC2 in Melanoma Cells. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 7614-7624.

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Transwell Boyden Chamber Invasion Assay

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Cell invasion was evaluated using the Transwell Boyden chamber system, following the protocol described by New et al. in 2017. In brief, 8-μm pore inserts were utilized for migration assessment. A thin layer of VitroGel was applied to the inserts. HNSCC cells (3 × 10⁴ cells/well) within serum-free culture media were plated on the VitroGel-coated inserts. These inserts were then set in triplicate containment wells containing media containing serum up to 24 hours. Simultaneously, cells (2 × 10³ cells/well in a 96-well plate) were plated under identical conditions to assess viability using the CyQUANT assay. Following fixative treatment then staining with the Hema 3 kit (Thermo Fisher Scientific), quantity of cells that invaded through the VitroGel and across the Boyden chamber membrane was quantified. The number of invading cells was normalized based on cell viability. To measure viability, HNSCC cells were seeded in quadruplicate (2 × 10³ cells/well in a 96-well plate) at initial day 0. Experimental conditions were applied on day 1 for durations of 24 to 72 hours, and cellular viability was quantified using a CyQUANT proliferation kit (Invitrogen C7026) pursuant to the manufacturer’s guidelines.

Arnold L., Yap M., Jackson L., Barry M., Ly T., Morrison A., Gomez J.P., Washburn M.P., Standing D., Yellapu N.K., Li L., Umar S., Anant S, & Thomas S.M. (2024). DCLK1-Mediated Regulation of Invadopodia Dynamics and Matrix Metalloproteinase Trafficking Drives Invasive Progression in Head and Neck Squamous Cell Carcinoma. bioRxiv.

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Analysis of BAL Cytokines and Mucins

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Screening Tumor Cell Invasion Using siRNA Knockdown

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siRNA pools targeting histone demethylases were purchased from Santa Cruz Biotechnology. MDA-MB-231 cells were transfected with siRNAs using Lipofectamine RNAiMax (Invitrogen). 48 hr post transfection, the knockdown efficiency was determined by Real-time RT-PCR. Primers used for the human histone demethylases were described previously.^{4 (link)} To screen tumor cell invasion, 1×10⁵ siRNA-transfected cells were seeded in triplicate on BD BioCoat Matrigel Invasion Chambers. After 24 hours, matrigel was removed, and the invaded cells were stained with the HEMA 3 kit (Fisher). The invaded cells were counted from at least 5 random fields and averaged.

Ramadoss S., Guo G, & Wang C.Y. (2016). Lysine demethylase KDM3A regulates breast cancer cell invasion and apoptosis by targeting histone and the non-histone protein p53. Oncogene, 36(1), 47-59.

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