Anti mouse cd45 efluor450

Manufactured by Thermo Fisher Scientific

The Anti-mouse CD45 eFluor450 is a fluorochrome-conjugated monoclonal antibody that specifically binds to the mouse CD45 cell surface antigen. CD45 is a receptor-linked protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells.

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6 protocols using anti mouse cd45 efluor450

Comprehensive Murine Immune Cell Analysis

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Mouse ears were subjected to enzymatic digestion as previously described (Feuerstein et al., 2015 (link)). After digestion, samples were filtered with a 70 µm cell strainer (BD), washed with FACS Buffer (PBS+2%FBS+2 mM EDTA) and stained with the indicated antibodies. The following antibodies were used: anti-mouse CD45 eFluor450 (eBioscience), anti-mouse CD11b PE-Cy7 (eBioscience), anti-mouse Ly6G FITC (BD Biosciences), anti-mouse Ly6C PerCP-Cy5.5 (BD Biosciences), anti-mouse CD64 PerCP/Cy5.5 (Biolegend), anti-mouse CD11c APC (BD Biosciences), mouse MHC class II eFluor450 (eBiosciences) and anti-mouse CD3e (Biolegend). Cell samples were analyzed with a 10-color flow cytometer (Gallios, Beckman Coulter) and the Kaluza software (version 1.5a, Beckman Coulter).

Feuerstein R., Forde A.J., Lohrmann F., Kolter J., Ramirez N.J., Zimmermann J., Gomez de Agüero M, & Henneke P. (2020). Resident macrophages acquire innate immune memory in staphylococcal skin infection. eLife, 9, e55602.

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Multicolor Flow Cytometry for Human Cell Engraftment in Humanized Mice

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To detect human cells engrafted in HUMAMICE, multi-colors cytometric analysis was performed using BD LSR Fortessa, according to the manufacturer’s protocol but with a minor modification. HUMAMICE were sacrificed and the spleens were removed at three different times after hPBMCs were transplanted. Splenocytes were collected and lysed by ACK lysis buffer, counted and incubated with an appropriate volume of antibodies for 1 hour at 4°C and then subjected to flow cytometry analysis.
Commonly used antibodies included: Anti-Human CD45 FITC, Clone: 2D1, (ebioscience9011‐9459); isotype control, Mouse IgG1 K Isotype Control (ebioscience, FITC 11–4714) ; Anti-Mouse CD45 eFluor^® 450 (ebioscience, 48-0451-82) ; isotype control, Mouse IgG2b K Isotype Control eFluor^® 450 (ebioscience, 48-4732-82) ; Anti-Human CD3 APC-eFluor^® 780, Clone: UCHT1, (ebioscience, 47–0038) ; Mouse IgG1 K Isotype Control APC- eFluor^® 780 (ebioscience, 47–4714) ; Anti-Human CD4 PerCP-Cyanine5.5, Clone: OKT4 (OKT-4), (ebioscience, 45–0048); isotype control, Mouse IgG2b K Isotype Control PerCP-Cyanine5.5 (cat. 45–4732) ; Anti-Human CD8a PE-Cyanine7, Clone: SK1, (ebioscience, 25–0087); isotype control, Mouse IgG1 K Isotype Control PE-Cyanine7 (ebioscience, 25–4714) ; Anti-Human CD19 APC, Clone: 2H7, (ebioscience, 17–0209); isotype control, Mouse IgG1 K Isotype Control APC (ebioscience, 17–4714).

Zeng Y., Liu B., Rubio M.T., Wang X., Ojcius D.M., Tang R., Durrbach A., Ru Z., Zhou Y, & Lone Y.C. (2017). Creation of an immunodeficient HLA-transgenic mouse (HUMAMICE) and functional validation of human immunity after transfer of HLA-matched human cells. PLoS ONE, 12(4), e0173754.

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Isolation and Characterization of Adipose Stromal Vascular Fraction

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The stromal vascular fraction (SVF) was isolated from whole adipose tissue as previously described (Cho, Morris and Lumeng, 2014 (link)). Briefly, adipose tissue depots were dissected and weighed. Tissue was then mechanically disrupted by mincing, and chemically digested by rocking tissue in 1mg/ml collagenase IV (Sigma Aldrich) at 37C for 30 mins. Cells were then quenched with RPMI media and filtered through 100nm mesh prior to RBC lysis and subsequent filtering with 70nm mesh filters.
Cells were incubated in Fc Block for 5 minutes on ice and stained with indicated antibodies for 30 minutes at 4C: Anti-Mouse CD45 eFluor 450 [48–0451-82], Anti-Mouse CD8a FITC [11–0081-82], Anti-Mouse MHC Class II (I-A/I-E) PE-Cy7 [25–5321-82], Anti-Mouse CD4 APC [17–0041-82], Anti-Mouse CD11c APC-eFluor® 780 [47–0114-80], from eBioscience, anti-mouse CD3ε PerCP/Cy5.5 [145–2C11] from Biolegend, and anti-Mouse CD64 a and b Alloantigens PE [558455] from BD Pharmingen Stained cells were washed twice with FACS buffer and fixed for intracellular staining with Anti-Mouse Foxp3 PE [12–4771-82] from eBioscience, using a FoxP3 transcription kit (BD Biosciences). Analysis was performed on a FACSCanto II Flow Cytometer and analyzed with Flow Jo software (Treestar).

Porsche C.E., Delproposto J.B., Patrick E., Zamarron B.F, & Lumeng C.N. (2020). Adipose tissue dendritic cell signals are required to maintain T cell homeostasis and obesity-induced expansion. Molecular and cellular endocrinology, 505, 110740.

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Quantifying Human Cell Engraftment

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Human engraftment was determined in bone marrow harvested from tibiae or femora. Human engraftment was monitored as the percentage of human cells over all human (anti-human-CD19 PE [BioLegend]; anti-human-CD7 PE [ebiosciences]; anti-human-CD45 Alexa Fluor 647 [BioLegend]) and murine leukocytes (anti-mouse-CD45 eFluor 450, [ebiosciences]) (Online Supplementary Methods).

Barz M.J., Behrmann L., Capron D., Zuchtriegel G., Steffen F.D., Kunz L., Zhang Y., Vermeerbergen I.J., Marovca B., Kirschmann M., Zech A., Nombela-Arrieta C., Ziegler U., Schroeder T., Bornhauser B, & Bourquin J.P. (2022). B- and T-cell acute lymphoblastic leukemias evade chemotherapy at distinct sites in the bone marrow. Haematologica, 108(5), 1244-1258.

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Phosphorylation of Stat1 in Immune Cells

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Mouse ears were subjected to enzymatic digestion, filtered, washed and cells finally resuspended in 100 µl RPMI medium supplemented with 10% FBS and antibiotics (ciprofloxacin, 10 mg/ml). Then cells were stimulated for 15 min at 37°C with 200 ng/ml IFNγ and subsequently fixed with 100 µl fixation buffer (Cytofix Fixation Buffer, BD Biosciences) for 10 min at 37°C. After centrifugation and permeabilization with 400 µl ice cold Perm Buffer III (BD Phosflow) for 30 min at 4 °C cells were washed twice with PBS containing 1% BSA. Then cell surfaces were stained with anti-mouse CD45 eFluor450 (eBioscience), anti-mouse CD11b PE-Cy7 (eBioscience), anti-mouse CD11c APC (BD Biosciences) and intracellular phosphorylated Stat1 stained with mouse anti-Stat1 (pY701) Alexa Fluor 647 (BD Biosciences) 30 min at RT and subsequently analyzed by flow cytometry.

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Wnt3a-Mediated Xenograft Transplantation

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This study was carried out in accordance to the Dutch law on Animal Welfare and Experiments. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Erasmus University Medical Centre Rotterdam, The Netherlands. Intrabone transplantations were performed under isoflurane anesthesia. All animals were housed in groups in individually ventilated cages. Food and water were available ad libitum.
NSG mice were sublethally irradiated (3 Gy) and subsequently transplanted with the progeny generated from 1,00E+05 UCB-derived CD34⁺ cells cultured in our SFT medium, with or without the addition of 250 ng/ml Wnt3a for 7 days. Each group contained 5 mice. Engraftment was assessed every 2 weeks starting at 3 weeks after transplantation by flowcytometric analysis of the peripheral blood, using a flowcytometric panel including anti-mouseCD45-eFluor450, (eBioscience, Vienna, Austria) and anti-humanCD45-APC-Cy7 (BioLegend, London, UK). Mice were considered engrafted when human CD45 levels were higher than 0.1%. At 17 weeks after transplantation, the mice were sacrificed by cervical dislocation and cells from femurs were analysed.

Duinhouwer L.E., Tüysüz N., Rombouts E.W., ter Borg M.N., Mastrobattista E., Spanholtz J., Cornelissen J.J., ten Berge D, & Braakman E. (2015). Wnt3a Protein Reduces Growth Factor-Driven Expansion of Human Hematopoietic Stem and Progenitor Cells in Serum-Free Cultures. PLoS ONE, 10(3), e0119086.

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