The following cell staining antibodies were purchased from Biolegend (San Diego, CA, USA): PC7-conjugated anti-mouse Gr-1 antibody, PC5-conjugated anti-mouse CD11b antibody, PE-conjugated anti-mouse CD40 antibody, FITC-conjugated anti-mouse CD45 antibody (Biolegend, San Diego, USA). PE-conjugated CXCR5 antibody was purchased from Miltenyi. Flow cytometry was performed using a Cytomics FC 500 flow cytometer (Beckman Coulter, Brea, CA USA).
Fitc conjugated anti mouse cd45 antibody
Manufactured by BioLegend
Sourced in United States
FITC-conjugated anti-mouse CD45 antibody is a laboratory reagent used to identify and quantify mouse CD45-positive cells in various sample types. It is a fluorescently labeled antibody that binds specifically to the CD45 surface antigen, which is expressed on most hematopoietic cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa cell analyzer, by BD (3 mentions)
Percp cy5.5 conjugated anti mouse f4 80 antibody, by BioLegend (2 mentions)
Cytomics fc500 flow cytometer, by Beckman Coulter (1 mentions)
Zombie violet dye, by BioLegend (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Hank s balanced salt solution, by Lonza (1 mentions)
Percp cyanine5.5 conjugated anti mouse cd8a antibody, by BioLegend (1 mentions)
Isotype control antibody, by BioLegend (1 mentions)
Hanks balanced salt solution (hbss), by Lonza (1 mentions)
70 μm cell sorters, by BD (1 mentions)
Cell recovery solution, by Corning (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
5 protocols using fitc conjugated anti mouse cd45 antibody
1
Multicolor Flow Cytometry Staining
2
Isolation and Analysis of Colonic Leukocytes
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Distal colon tissues were dissected and digested with Hank's-balanced salt solution (Lonza) supplemented with 1 mM dithiothreitol (DTT) and 5 mM EDTA overnight at 4 °C. After filtering through 70 μm cell strainer (BD Biosciences), the single-cell suspensions were stained with FITC-conjugated anti-mouse CD45 antibody, PerCP/Cy5.5-conjugated anti-mouse F4/80 antibody, PE/Cy7-conjugated anti-mouse Ly-6G/Ly-6C (GR-1) antibody, isotype control antibody and Zombie Violet™ dye (BioLegend). Data were acquired using BD LSRFortessa™ cell analyzer (BD Biosciences) and analyzed using FlowJo software (FlowJo LLC). In our analysis, leukocytes were identified as CD45+ cells, macrophages were identifed as CD45+ F4/80+ cells, and neutrophils were identified as CD45+ GR-1+ cells.
3
Tumor Immune Cell Characterization
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The mice shown in Figure 5 were sacrificed on Day 14 after a total of six doses, and the tumor tissues were recovered. Tissues were first cut into small pieces with scissors and washed with PBS. Then, collagenase/hyaluronidase was added to digest the tissues with supplement of DNase I. The digestion proceeded at 37°C with continuous shaking for 30 min before quenching with DMEM supplemented with FBS. The digested suspension then flowed through a cell strainer (100 μm). The recovered cells were washed and resuspended in PBS. Cells were first stained with a Zombie NIR™ Fixable Viability Kit (catalog no. 423106) and blocked with TruStain fcX™ anti‐mouse CD16/32 (catalog no. 101320). Subsequently, the cells were stained with FITC‐conjugated anti‐mouse CD45 antibody (Biolegend, catalog no. 103108) and PerCP/Cyanine5.5‐conjugated anti‐mouse CD8a antibody (Biolegend, catalog no. 100734). The stained cells were washed with PBS and ready for flow cytometry analysis after resuspension in PBS. Single and live cells with surface CD45 and CD8 markers were gated and quantitatively analyzed.
4
Isolation and Analysis of Colon Immune Cells
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The distal colon tissues from the mice were dissected, washed with cold PBS, and digested with Hank’s-balanced salt solution (HBSS, Lonza, Basel, Switzerland) supplemented with 1 mM dithiothreitol (DTT) and 5 mM ethylenediaminetetraacetic acid (EDTA) for 2 h at 4°C. The single-cell suspensions were filtered through 70 μm cell filters (BD Biosciences, San Jose, CA). The cells were stained with FITC-conjugated anti-mouse CD45 antibody, PerCP/Cy5.5-conjugated anti-mouse F4/80 antibody, and isotype control antibody according to the manufacturer’s instructions (BioLegend, San Diego, CA). The stained cells were analyzed using BD LSRFortessa™ cell analyzer (BD Biosciences, San Jose, CA) and data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR). In our analysis, leukocytes were identified as CD45+ cells and macrophages were identified as CD45+ F4/80+ cells.
5
Matrigel Plug Angiogenesis Assay
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Animal experiments were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of University of Massachusetts Amherst. Briefly, 0.25 mL growth factor-reduced Matrigel (BD Biosciences, San Jose, CA), which was pre-mixed with mutanocyclin or DMSO vehicle, was subcutaneously injected into 6-week-old C57BL/6 male mice in the abdominal area. After 5 days, the mice were euthanized to dissect the implanted Matrigel plugs. The plugs were digested using Corning® cell recovery solution (Corning, NY), filtered through 70 μm cell sorters (BD Biosciences, San Jose, CA) to obtain single cell suspension, which were stained with FITC-conjugated anti-mouse CD45 antibody (catalog number: 103107; clone: 30-F11, dilution ratio: 1:500) and isotype control antibody (catalog number: 400605; clone: RTK4530, dilution ratio: 1:500, BioLegend, San Diego, CA). The stained cells were analyzed using BD LSRFortessa™ cell analyzer (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR). Gating and cell identification strategies are as follows: cell doublets and clumps were eliminated using FSC-H vs. FSC-A gating, and debris was eliminated using FSC-A vs. SSC-A. Dead cells were gated out using Zombie Violet™ dye. In our analyses, leukocytes were identified as CD45+ cells.
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