Cd45 pacific blue

Manufactured by Thermo Fisher Scientific

Sourced in United States

The CD45 Pacific Blue is a flow cytometry reagent used for the identification and enumeration of CD45-positive cells. It is a fluorescently labeled antibody that binds to the CD45 antigen, which is expressed on the surface of most hematopoietic cells. The Pacific Blue fluorochrome provides a bright signal that can be detected using standard flow cytometry instrumentation.

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Anti-CD45-Pacific Blue (3 citations)

5 protocols using cd45 pacific blue

Delineate Innate Lymphoid and Macrophage Subsets

Cited 2 times

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Stained cells underwent analysis utilizing FACS Canto II, and the resultant data were processed utilizing FlowJo version 10 software (Ashland, OR, USA). The following antibodies procured from eBioscience (Thermo Fisher Scientific, Waltham, MA, USA) were employed for the delineation of innate lymphoid cells: Biotin-CD3e (100304; clone: 145-2C11; 1/200), Biotin-CD45R/B220 (103204; clone: RA3–6B2; 1/200), Biotin-Gr-1 (108404; clone: RB6-8C5; 1/200), Biotin-CD11c (117304; clone: N418; 1/200), Biotin-CD11b (101204; clone: M1/70; 1/200), Biotin-Ter119 (116204; clone: TER-119; 1/200), Biotin-FceRIa (134304; clone: MAR-1; 1/200), FITC-Streptavidin (405202; 1/500), PE-Cy7-CD127 (135014; clone: A7R34; 1/100), Pacific Blue-CD45 (103116; clone: 30-F11; 1/100), PE-GATA-3 (clone: TWAJ; 1/50), APC-RORγ (clone: AFKJS-9; 1/50), and Fixable Viability Dye eFluor 780 (1/400) [20 (link),21 (link)]. Additionally, the following antibodies (eBioscience, San Diego, CA, USA) were utilized for the discrimination of M1 and M2 macrophages: APC-CD45.2 (17045482; clone: 104; 1/50), PE-F4/80 (12480182; clone: BM8; 1/50), APC-Cy7-CD11b (47011282; clone: M1/70; 1/50), FITC-CD206 (MA516870; clone: MR5D3; 1/50), and PE-Cy7-CD11c (25011482; clone: N418; 1/50) [22 (link)].

Hosomi Y., Okamura T., Sakai K., Yuge H., Yoshimura T., Majima S., Okada H., Senmaru T., Ushigome E., Nakanishi N., Satoh T., Akira S., Hamaguchi M, & Fukui M. (2024). IL-33 Reduces Saturated Fatty Acid Accumulation in Mouse Atherosclerotic Foci. Nutrients, 16(8), 1195.

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Multiparametric flow cytometry analysis

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For surface marker staining, mononuclear cells were previously blocked with anti-CD16/32 antibody (Thermo Fisher Scientific) for 15 min and then incubated with the following anti-mouse antibodies: PE-CD19, PE-B220 and Pacific blue-CD45 (all purchased from Thermo Fisher Scientific).
For intracellular marker staining, cells were previously stimulated with 50 ng/mL PMA and 750 ng/mL ionomycin in the presence of 20 µg/mL brefeldin A for 5 h, then surface-stained for 30 min. After surface marker staining, cells were resuspended in Fixation/Permeabilization Buffer Set (Thermo Fisher Scientific) and stained with PE-Cy7-IL-10 (BioLegend, San Diego, CA, USA); APC-IL-12A (Thermo Fisher Scientific) and FITC-EBI3 (Novus Biologicals, Littleton, CO, USA) for 45 min according to the manufacturer’s protocol.
For intranuclear (p-STAT3) marker staining, cells were previously surface stained and then fixed and permeabilized using a transcription factor staining buffer set (Thermo Fisher Scientific) and stained with PE-p-STAT3 (Thermo Fisher Scientific) for 45 min according to the manufacturer’s protocol.
Isotype antibodies were used as negative controls. Flow cytometry analysis was performed using BD Celesta Analyzer.

Xie M., Zhu Y., Zhou Y., Wang Q., Gu E., Chu Y, & Wang L. (2023). Interleukin-35 -producing B cells rescues inflammatory bowel disease in a mouse model via STAT3 phosphorylation and intestinal microbiota modification. Cell Death Discovery, 9, 67.

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Adipose Tissue Macrophage Analysis

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Mice were euthanized and adipose tissue depots and liver were dissected at the end of the experiment. Adipose tissue was subjected to collagenase digestion to isolate the stromal vascular fraction (SVF) as described^{30 (link)}. SVF was incubated with antibodies to identify macrophage subsets and proliferation and analyzed by flow cytometry on the BD Biosciences LSRFortessa™ cell analyzer as described^{30 (link)}. The following conjugated antibodies were used: CD301 APC (Clone ER-MP23, BioRad, Cat# MCA2392A647), CD45 Pacific Blue (Clone 30-F11, eBiosciences, Cat# 48,045,182), CD64 PE (Clone X54-6/7.1.1, BD Biosciences, Cat# 558,455), CD11c APC-Cy7 (Clone N418, eBiosciences, Cat# 47,011,482), and Ki67 PeCy7 (Clone SolA15, eBiosciences, Cat#25,569,882). Single-stained, isotype, and unstained SVF were used as controls.

Nance S.A., Muir L., Delproprosto J, & Lumeng C.N. (2023). MSR1 is not required for obesity-associated inflammation and insulin resistance in mice. Scientific Reports, 13, 2651.

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Assay for Platelet-Leukocyte Aggregates

Cited 1 time

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ACD-whole blood collected from the retro-orbital plexus was incubated with an excess of ice-cold lysis buffer plus 1 μM prostaglandin E1 (PGE₁) to prevent PLT activation and in vitro formation of PLAs as described.^{29 (link)} The blood cells were lysed briefly (1 min), spun at 4°C and stained with a cocktail of antibodies at 1:100 dilution: CD45-Pacific blue (eBioscience), CD115-APC (eBioscience), Gr-1-PerCP cy5.5 (Ly6-C/G; BD Biosciences), CD11b-PE Cy7 (eBioscience) and CD41-FITC (eBioscience). Platelet Ly-6C^hi monocyte aggregates, platelet Ly-6C^lo monocyte aggregates and platelet neutrophil aggregates were defined as CD45⁺Gr-1^hiCD115⁺CD41⁺, CD45⁺Gr-1^loCD115⁺CD41⁺ and CD45⁺Gr-1⁺CD115⁻CD41⁺.

Wang W., Oh S., Koester M., Abramowicz S., Wang N., Tall A.R, & Welch C.L. (2016). Enhanced Megakaryopoiesis and Platelet Activity in Hypercholesterolemic, B6-Ldlr−/−, Cdkn2a-Deficient Mice. Circulation. Cardiovascular genetics, 9(3), 213-222.

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Multiparameter Flow Cytometry Immunophenotyping

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The following conjugated Anti-human monoclonal antibodies (mAbs) were purchased from BD Pharmingen, USA: CD3 PE-Cy7, CD4 FITC, CD4 PerCP-Cy5.5, CD4 APC, CD8 PerCP-Cy5.5, CD14 PErCP-Cy5.5, CD14 PE, CD80 FITC, CD86 PE, CD152 PE, HLA-DR APC, Ki67 PE, Annexin V FITC, and Propidium Iodide. CD28 FITC and CD45 Pacific blue were purchased from eBioscience, USA.

Kumar G., Cottalorda-Dufayard J., Garraffo R., De Salvador-Guillouët F., Cua E, & Roger P.M. (2022). Raltegravir Inclusion Decreases CD4 T-Cells Intra-Cellular Viral Load and Increases CD4 and CD28 Positive T-Cells in Selected HIV Patients. Cells, 11(2), 208.

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