Cd45 pe cy5

Manufactured by BioLegend

Sourced in Germany

CD45-PE/Cy5 is a fluorescently-labeled antibody that binds to the CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of most hematopoietic cells. The PE/Cy5 conjugate allows for detection of CD45-positive cells using flow cytometry.

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8 protocols using cd45 pe cy5

Flow Cytometry Technique for Murine Blood

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Two hundred microliters of facial blood were obtained from a mouse and mixed with 20μl of 3.8% sodium citrate. The blood/sodium citrate mixture was then fixed in 1% paraformaldehyde (PFA) at room temperature for 10 min. Following fixation 8 ml of red blood cell lysing buffer (Sigma) were added and incubated on ice for 10 min. After incubation, 1 ml of 10× DPBS (Invitrogen) was added and mixed thoroughly then centrifuged at 1,800×g for 2 min. The pellet was then resuspended in 1× DPBS. The white blood cells were then blocked with rat whole IgG (Jackson ImmunoResearch) and hamster whole IgG (Biolegend) at 4ºC for 5 min. After blocking the following antibodies were used to identify interactions: PE/Cy5-CD45 (Biolegend), PE-CD11c (Biolegend), FITC-CD41 (BD Biosciences). For controls the following isotype-matched antibodies were used: PE/Cy5-Rat IgG_2b (Biolegend), PE-Hamster IgG_1κ (Biolegend) and FITC-Rat IgG₁ (Biolegend). The mixtures of antibodies and white blood cells were then incubated on a rotator for 30 min at room temperature and then measured with a Coulter Epics XL-MCL Flow Cytometer and analyzedCoulter Epics XL-MCL) Coulter Epics XL-MCL) using EXPO32 ADC software (Beckman Coulter).

Zhou H., Ran Y., Da Q., Shaw T.S., Shang D., Reddy A.K., López J.A., Ballantyne C.M., Ware J., Wu H, & Peng Y. (2016). Defective association of the platelet glycoprotein Ib-IX complex with the glycosphingolipid-enriched membrane domain inhibits murine thrombus and atheroma formation. Journal of immunology (Baltimore, Md. : 1950), 197(1), 288-295.

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Isolation and Purification of Viable Hepatocytes

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Viable hepatocytes were collected using a method adapted from Ben-Moshe et al. (2019) (link). In details, isolated hepatocytes were resuspended in 25mL EBSS then mixed with 22.5ml Percoll (Sigma) and 2.5mL 10xPBS to remove dead cells. The mixture was centrifuged at 600rpm for 10 minutes, supernatant was discarded, and the cells were resuspended in cold FACS buffer (2mM EDTA, 10% FBS in 1x PBS). 100μl of 10⁶ cells were aliquot into FACS tubes and .25μg FcX blocking solution (Biolegend) was added. Cells were incubated on ice for 30 minutes. Next, 2ml of FACS buffer was added to each tube, and centrifuged at 1,000rpm for 3 minutes to wash the cells. Cells were resuspended in 100μl FACS buffer and stained with APC-CD31(BioLegend, cat: 102509), PE/Cy5-CD45 (BioLegend, cat: 103109), APC/Cy7-CXCR2 (BioLegend, cat: 149313) in a dilution of 1:100.

Nguyen N.T., Umbaugh D.S., Sanchez-Guerrero G., Ramachandran A, & Jaeschke H. (2021). Kupffer cells Regulate Liver Recovery Through Induction of Chemokine Receptor CXCR2 on Hepatocytes after Acetaminophen Overdose in Mice. Archives of toxicology, 96(1), 305-320.

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Multiparametric Immunophenotyping of BMMCs

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BMMC were stained with the following anti-human antibodies: IgD-Brilliant Violet 480 (Cat. #566138; BD Biosciences), CD3-BUV737 (Cat. #612750; BD Biosciences), CD14-BUV737 (Cat. #612763; BD Biosciences), CD19-Spark NIR 685 (Cat. #302270; BioLegend), CD38-Brilliant Violet 785 (Cat. #303530; BioLegend), CD138-APC-R700 (Cat. #566050; BD Biosciences), CD27-Brilliant Violet 711 (Cat. #356430; BioLegend), CD134 (OX40)-Brilliant Violet 510 (Cat. #350025; BioLegend), CD246 (ALK)-Alexa Fluor 488 (Cat. #NBP3–08771AF488; Novus), CD357 (GITR)-Brilliant Violet 605 (Cat. #747664; BD Biosciences), and CD45-PE-Cy5 (Cat. #304009; BioLegend). Samples were run on a Cytek’s Aurora Spectral Flow Cytometer analyzed with the FlowJo v10.8.1 software (FlowJo, LLC).

Lee F., Nguyen D., Hentenaar I., Morrison-Porter A., Solano D., Haddad N., Castrillon C., Lamothe P., Andrews J., Roberts D., Lonial S, & Sanz I. (2024). The Majority of SARS-CoV-2 Plasma Cells are Excluded from the Bone Marrow Long-Lived Compartment 33 Months after mRNA Vaccination. Research Square.

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Comprehensive Testis Immune Profiling

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The immune infiltration in the testis was analyzed as previously described.^{20 (link)} Briefly, testis tissues were collected, mechanically dissociated, and then digested in PBS containing 1 mg/mL Collagenase IV/0.15 mg/mL DNase I for 40 min at 37°C. After passing through a nylon mesh, cells were blocked with anti-CD16/32 antibody and then stained with conjugated antibodies to CD4-FITC, CD8a-APC, CD45-PE/Cy5, CD25-APC, CD117(c-kit)-APC, CD274(B7-H1)-PE, FcεRIα-FITC, CD34-PE, or FOXP3-PE (all from BioLegend). For intracellular staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). The samples were assessed with BD Calibur flow cytometry, and the data were analyzed with FlowJo software (V. 10.6.2, Tree Star).

Wang L., Jiang G., Jing N., Liu X., Zhuang H., Zeng W., Liang W, & Liu Z. (2021). Downregulating testosterone levels enhance immunotherapy efficiency. Oncoimmunology, 10(1), 1981570.

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Characterizing Macrophage Response to Kidney Injury

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To determine the potential effects of HU and kidney injury on macrophage numbers and phenotype, kidneys were harvested on day 3 and 14 post IR injury. Single-cell suspensions were prepared using collagenase/DNase1 solution for 45 min at 37 °C. After centrifugation (1500 rpm for 5 min 4 °C, without break), cells were stained with the Live/Dead dye (Zombie NIR). Cells were resuspended in 1 mL PBS containing 0.5% BSA and 0.2 mM EDTA. Unspecific binding was blocked using FcR block (CD16/32) [36 (link)]. After washing, cells were stained with anti-mouse macrophage/myeloid cells markers CD45-PE/Cy5, F4/80-APC, CD206-FITC, CX3CR1-PE, MHCII-V450 (all from BioLegend, Fell, Germany), and anti-mouse CD11b-BV510 (BD Bioscience, Heidelberg, Germany). M2-like macrophages were identified as CD45 + CD11b + F4/80 + CX3CR1 + CD206+, whereas M1-like macrophages as CD45 + CD11b + F4/80 + CX3CR1 + CD206- [31 (link)]. Flow cytometry was performed on the BD FACSCanto II (BD, NJ, USA), and the data were analyzed using FlowJo 8.7 software (Tree Star, Ashland, OR, USA).

Gnemmi V., Li Q., Ma Q., De Chiara L., Carangelo G., Li C., Molina-Van den Bosch M., Romagnani P., Anders H.J, & Steiger S. (2022). Asymptomatic Hyperuricemia Promotes Recovery from Ischemic Organ Injury by Modulating the Phenotype of Macrophages. Cells, 11(4), 626.

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Immunophenotyping and Protein Expression

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Most primary and secondary antibodies came from Abcam: mouse monoclonal CXCL12 (MM0211-9N26), rabbit recombinant monoclonal SCF (EP665Y), rabbit polyclonal HIF-1α (AB103063), or mouse monoclonal connexin-43 (Cx-43) (4E6.2). Goat Anti-Mouse Alexa Fluor^® 488 (IgG H&L) (ab150113), Goat Anti-Rabbit Alexa Fluor^® 405 (IgG H&L) (ab175652), and Donkey Anti-Mouse Alexa Fluor^® 405 (IgG H&L) (ab175658). Rabbit Anti-Human phospho-NF-kB p65 (ser536) (93H1) came from cell signaling. Source for immunophenotyping antibodies: CD34-APC (Clone 581, BioLegend), CD19-FITC (Clone HIB19, BioLegend), CD45-PECy5 (Clone HI30, BioLegend), HLA-DR-PE (clone L243, BioLegend), CD90-APC (5E10, BD), CD73-PE (AD2, BD), and CD105-FITC (266, BD).

Balandrán J.C., Purizaca J., Enciso J., Dozal D., Sandoval A., Jiménez-Hernández E., Alemán-Lazarini L., Perez-Koldenkova V., Quintela-Núñez del Prado H., Rios de los Ríos J., Mayani H., Ortiz-Navarrete V., Guzman M.L, & Pelayo R. (2017). Pro-inflammatory-Related Loss of CXCL12 Niche Promotes Acute Lymphoblastic Leukemic Progression at the Expense of Normal Lymphopoiesis. Frontiers in Immunology, 7, 666.

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Murine Hematopoietic Stem Cell Analysis

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For bone marrow, cells were stained with antibodies to (hybridoma names and vendors in parentheses): Kit-APC/Cy7 (2B8, Becton Dickinson), CD27-FITC (LG.3A10, Biolegend), CD201-APC (RCR-16, Biolegend), Sca-1 (D7, BV605, BD; and E13-161-7, Pacific Blue, Biolegend), CD150-Biotin (TC15-12F12.2; with streptavidin-Brilliant Violet 421, BD), CD4 (GK1.5), CD8 (53-6.7), CD3ε (145-2C11), Ter119 (TER-119), B220 (RA3-6B2), Gr1 (RB6-8C5), Mac-1 (M1/70) were used. Antibodies to CD4, CD8, CD3ε, Ter119, B220, Gr-1 and Mac-1 were purchased as conjugates to PE/Cy5 (Biolegend). For peripheral blood, cells were stained with antibodies to: CD3ε-Pacific Blue (17A2, Biolegend), CD3ε-PE (145-2C11, Biolegend) CD45-PECy5 (30-F11, Biolegend), Mac-1-PECy7 (M1/70, Biolegend), Gr1-Alexa Fluor 680 (in house), B220-Pacific Blue (RA3-6B2, Biolegend), B220-FITC (in house). Cells were stained on ice in staining buffer for 1 hour, washed, and resuspended in staining buffer with propidium iodide (0.25ug/ml). Analysis and sorting of stained cells were performed on an LSRII (BD Biosciences) or on a FACSAria (BD Biosciences) at the Joslin Diabetes Center Flow Core.

Vazquez S.E., Inlay M.A, & Serwold T. (2015). CD201 and CD27 identify hematopoietic stem and progenitor cells across multiple murine strains independently of Kit and Sca-1. Experimental hematology, 43(7), 578-585.

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Corneal Immune Cell Profiling

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Mouse eyeballs were collected and fixed in PBS at 48 h or 72 h after injury. Cornea was dissected around the scleral-limbal region and cut into pieces by corneal scissors. We incubated them in 50 uL Liberase TL (2.5 mg/ml; Sigma) for 30 min at 37 °C with gentle stirring and then undigested tissue removed by screen filters (4 corneas were pooled as a sample). We finally collected cell suspensions and incubated them with fluorescein-conjugated anti-mouse antibodies for 30 min at 4 °C and analyzed by flow cytometry with a Vantage flow cytometer (Becton Dickinson) and FlowJo (Tree Star, Inc.) software. The gate was set on CD45+ population, and further analysis of surface markers was done with in this gate. The primary antibodies used were as follows: CD45-PE/-cy5, CD86-PE-cy5, F4/80-FITC, CD11b- FITC, Ly6G-PE and CD206-PE (Biolegend, San Diego, CA). All experiments were repeated at least 3 times.

Zhang Y., Chen P., Di G., Qi X., Zhou Q, & Gao H. (2018). Netrin-1 promotes diabetic corneal wound healing through molecular mechanisms mediated via the adenosine 2B receptor. Scientific Reports, 8, 5994.

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