Anti cd45 bv605

Manufactured by BD

Anti-CD45 BV605 is a fluorochrome-conjugated monoclonal antibody designed to detect the CD45 antigen expressed on the surface of various hematopoietic cells.

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4 protocols using anti cd45 bv605

Identification of Tumor-Infiltrating Immune Cells

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Tumor cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described by Rossowska et al. (30 (link)). Briefly, tumor-derived cells were stained with LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher) and then stained with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 AlexaFluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD86 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE, anti-CD44 PE-Cy7, anti-CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using the FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).

Rossowska J., Anger N., Wegierek K., Szczygieł A., Mierzejewska J., Milczarek M., Szermer-Olearnik B, & Pajtasz-Piasecka E. (2019). Antitumor Potential of Extracellular Vesicles Released by Genetically Modified Murine Colon Carcinoma Cells With Overexpression of Interleukin-12 and shRNA for TGF-β1. Frontiers in Immunology, 10, 211.

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Comprehensive Immune Profiling of Tumor and Spleen Cells

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Tumor cells and spleen cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described previously (46 (link)). Briefly, tumor single-cell suspensions were stained with the LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher Scientific, Inc.) and then labelled with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 Alexa Fluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD80 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE (all from BioLegend) for lymphocyte identification. Then, the cells were fixed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. In spleen single cell suspension only the lymphocyte identification was performed according to the procedure described above. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).

Szczygieł A., Anger-Góra N., Węgierek-Ciura K., Mierzejewska J., Rossowska J., Goszczyński T.M., Świtalska M, & Pajtasz-Piasecka E. (2021). Immunomodulatory potential of anticancer therapy composed of methotrexate nanoconjugate and dendritic cell-based vaccines in murine colon carcinoma. Oncology Reports, 45(3), 945-962.

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Immunophenotyping of Tumor-Infiltrating Cells

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Tumor samples were digested using half the amount of reagent required per sample of Miltenyi Biotech Liver Kit as per the manufacturer's instructions in C-tubes on a gentleMACS™ dissociator (Miltenyi Biotech) with heaters. After digestion, samples were filtered through Falcon filter cap tubes and washed with 2 ml Dulbecco's modified Eagle medium (Gibco). Samples were centrifuged at 500 g for 5 min, washed twice with AutoMACS Rinsing Solution (Miltenyi Biotech) with 1% bovine serum albumin and 0.5 mM EDTA, and resuspended in 1 ml of the aforementioned solution for counting and viability check on an Attune NxT cytometer. Cells (10⁶) were incubated with the following antibodies: anti-CD16/32 APC-R700, anti-CD11b APC, anti-F4/80 PE-Cy7, anti-EpCAM PerCP-Cy5.5, anti-CD45 BV605, anti-CD11c FITC, anti-NKp46 PE (BD Biosciences; diluted at 1:000 to 1:10.000). Data were acquired on a BD Biosciences FACSymphony A5 cytometer and analyzed with FlowJo Software (BD Biosciences).

Sargent J.K., Warner M.A., Low B.E., Schott W.H., Hoffert T., Coleman D., Woo X.Y., Sheridan T., Erattupuzha S., Henrich P.P., Philip V.M., Chuang J.H., Wiles M.V, & Hasham M.G. (2022). Genetically diverse mouse platform to xenograft cancer cells. Disease Models & Mechanisms, 15(9), dmm049457.

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Multicolor Flow Cytometry Assay for Murine Immune Cells

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For surface staining, single-cell suspensions were prepared as described before. Staining was performed with Live-Dead viability-staining (propidium iodide or 7-Aminoactinomycin 7AAD (BioLegend) or fixable viability stain 450 (BD Biosciences)). For analysis of the murine cell phenotype, the following antibodies were used:
Anti-CD4-V450 (1:400) (clone: RM4-5, BD Bioscience), Anti-CD4-PeCy7 (1:1000) (clone: RM4-5, BD Bioscience), Anti-CD4-AF700 (1:200) (clone: RM4-5, BD Biosciences), Anti-CD3-APC (1:200) (clone: 1452-C11, BD Bioscience), Anti-IFNγ-V450 (1:200) (clone: XMG1.2, BD Bioscience), Anti-CD45.2- AlexaFluor 700 (1:400) (clone: 104, Thermo Fisher Scientific), Anti-CD45-BV605 (1:200) (clone: 30-F11, BD Bioscience); Anti-CD11b- Pe/Cy7 (1:200) (clone: M1/70, Thermo Fisher Scientific), Anti-CD11b-PerCP-Cy5.5 (1:200) (clone: M1/70, Biolegend), Anti-IL 17-APC (1:200) (clone: eBio17B7, Thermo Fisher Scientific), Anti-CXCR5-FITC (1:200) (clone: L138D7, BioLegend) and Anti-PD-1-PE (1:200) (clone: 29F.1A12, BioLegend), Anti-Bcl6-PECy7 (1:50) (clone: 7D1, Biolegend), Anti-IL21-APC (1:50) (clone: FFA21, Thermo Fisher Scientific).
Flow cytometry was performed with a FACS Canto II^{57 (link)} and data were analyzed using FlowJo 10.0 software (https://www.flowjo.com/solutions/flowjo).

Baniahmad A., Birkner K., Görg J., Loos J., Zipp F., Wasser B, & Bittner S. (2020). The frequency of follicular T helper cells differs in acute and chronic neuroinflammation. Scientific Reports, 10, 20485.

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