Cd115 apc

Manufactured by BioLegend

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CD115-APC is a fluorescently labeled antibody that binds to the CD115 surface antigen. CD115 is a receptor tyrosine kinase that plays a role in the differentiation and function of monocytes and macrophages. The APC fluorescent label allows for the detection of cells expressing CD115 using flow cytometry or other fluorescence-based techniques.

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10 protocols using cd115 apc

Multicolor Flow Cytometry of Leukocytes

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To characterise circulating leukocytes by flow cytometry, 10 µL of heparinised whole blood was incubated for 30 min at RT with CD45-FITC (BD Biosciences, Franklin Lakes, NJ, USA) to identify leukocytes. Lymphocytes and neutrophils were identified by complexity and morphology, whereas monocytes were gated based on the CD115-APC marker (Biolegend, San Diego, CA, USA). Ly6C-PerCP (BD Biosciences, San Diego, CA, USA) and CD115-APC (Biolegend, San Diego, CA, USA) were used for Ly6C^low and Ly6C^hi-monocyte subsets identification. For circulating lymphocytes analysis, 10 µL of heparinised whole blood was incubated with 5 µL Brilliant Stain Buffer (BD Biosciences, San Diego, CA, USA) followed by CD4-BV421, CD8a-BV510, CD3e-APC, or CD69-PE antibodies (BD Biosciences, San Diego, CA, USA). Mouse Tregs were identified using Kit FoxP3 Staining Buffer Set and with anti-CD25-APC, anti-Foxp3-PE, and anti-CD4-VioBlue (all from Miltenyi, Bergisch Gladbach, Germany). The samples were incubated with FACS Lysing solution (BD Biosciences) for 10 min before flow cytometry analysis (FACSVerse or FACS Fortessa Flow cytometer, BD Biosciences, San Diego, CA, USA).

Taberner-Cortés A., Vinué Á., Herrero-Cervera A., Aguilar-Ballester M., Real J.T., Burks D.J., Martínez-Hervás S, & González-Navarro H. (2020). Dapagliflozin Does Not Modulate Atherosclerosis in Mice with Insulin Resistance. International Journal of Molecular Sciences, 21(23), 9216.

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Tracking Monocyte Recruitment in Atherosclerosis

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Circulating monocytes were labeled with 5-ethynyl-2′-deoxyuridine (EdU) to examine monocyte recruitment into atherosclerotic lesions, as described previously (35 (link)). Mice were injected intraperitoneally with 250 μl containing 1 μM EdU in saline (Invitrogen, Waltham, MA) two days before the sacrifice. To measure monocyte labeling efficiency, we examined EdU+ monocytes in circulation 48 h after injection by flow cytometry. We incubated blood samples with red blood cell lysis buffer (BioLegend, San Diego, CA) and stained them with primary antibodies antimouse CD45-FITC (Clone 30-F11; BioLegend), CD115-APC (Clone AFS98; BioLegend), and Ly6c-PerCP/Cy5.5 (Clone RB6-8C5; BioLegend) to identify circulating monocytes. Cells were permeabilized, and EdU was detected using Click-IT EdU Pacific Blue Flow Cytometry Assay Kit (Invitrogen). Flow cytometry was performed in LSR II analyzer (B.D. Biosciences, Franklin Lakes, NJ), and data were analyzed using FCS Express 5.01.0082 (De Novo Software, Pasadena, CA; https://denovosoftware.com/).

McQueen P., Molina D., Pinos I., Krug S., Taylor A.J., LaFrano M.R., Kane M.A, & Amengual J. (2024). Finasteride delays atherosclerosis progression in mice and is associated with a reduction in plasma cholesterol in men. Journal of Lipid Research, 65(3), 100507.

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Bone Marrow Macrophage Depletion

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Mice were injected with 250 μL of clodronate or PBS liposomes (ClodLip BV) intravenously at 9 days before tissue collection. BM macrophage depletion was confirmed using F4/80-FITC (Biolegend, 123108), Gr1-PE (Biolegend, 108408), and CD115-APC (Biolegend, 17-1152-82) antibodies.

Karatepe K., Zhu H., Zhang X., Guo R., Kambara H., Loison F., Liu P., Yu H., Ren Q., Luo X., Manis J., Cheng T., Ma F., Xu Y, & Luo H.R. (2018). Proteinase 3 Limits the Number of Hematopoietic Stem and Progenitor Cells in Murine Bone Marrow. Stem Cell Reports, 11(5), 1092-1105.

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Zinc Status of Murine Neutrophils

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Peripheral blood was collected into heparinized tubes from mice by cardiac puncture. Red blood cells were lysed for 5 min at 37°C using mouse red cell lysis buffer, and cells were then extensively washed and then resuspended at 4 × 10⁶ cells.mL^-1 in PBS with 1% BSA. To determine the Zn status of PMNs, cells were labelled with Fluozin-3 AM (Molecular Probes) for 30 min at 37°C and washed in PBS with 1% BSA. Following this, Fc receptors were blocked by incubation with 400 μg.mL^-1 murine gamma globulin (Rockland) for 15 min on ice before addition of fluorochrome conjugated monoclonal antibodies (CD115-APC, CD11b-BV421, Ly-6C-BV510, CD45-PE/Cy7 [Biolegend] and Gr1 (PE) [BD Biosciences]) and fixable live/dead reagent (Molecular Probes). Cells were stained for 30 min on ice in the dark, washed twice in cold PBS and then fixed in PBS with 1% PFA before analysis. Cells were acquired on a BD FACSAria and analysed using Flowjo software using the gating strategy shown in S8 Fig. PMNs were identified as live CD45⁺CD11b⁺Ly6C^intGr-1^hi. The fluorescence intensity of these cells with respect to Fluozin-3 staining was quantified.

Eijkelkamp B.A., Morey J.R., Neville S.L., Tan A., Pederick V.G., Cole N., Singh P.P., Ong C.L., Gonzalez de Vega R., Clases D., Cunningham B.A., Hughes C.E., Comerford I., Brazel E.B., Whittall J.J., Plumptre C.D., McColl S.R., Paton J.C., McEwan A.G., Doble P.A, & McDevitt C.A. (2019). Dietary zinc and the control of Streptococcus pneumoniae infection. PLoS Pathogens, 15(8), e1007957.

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Murine Blood Immune Profiling

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Mice were euthanized with CO₂ asphyxiation. Blood was recovered by hepatic portal vein puncture and collected in tubes containing 35 µl of 0.1 M EDTA. Whole blood (400 µl) was removed to a new tube, centrifuged at 2000g for 15 min, and plasma was removed and stored at −20°C. Cells were subjected to ammonium chloride erythrocyte lysis, washed with PBS, and stained with eFluor 718 fixable viability dye (eBioscience) according to the manufacturer’s instructions. Cells were then briefly fixed for 30 min in 0.1% PFA, washed, and Fc receptors were blocked with TruStain FcX (BioLegend) in 5% FBS overnight at 4°C. Cells were then stained with the following antibody mixtures for 30 min on ice: CD4–phycoerythrin (PE) (RM4-4; BioLegend), CD8- PerCP/eFluor 710 (H35-17.2; eBioscience), CD19–eFluor 450 (ID3; eBioscience), CD11b–APC–eFluor 780 (M1/70; eBioscience), Ly6C–FITC (fluorescein isothiocyanate) (HK1.4; Southern Biotechnology), Ly6G-PECy7 (1A8; BioLegend), and CD115-APC (AFS98; BioLegend). Liquid counting beads (BD) were added to each sample, and data were obtained on a BD LSR II (Becton Dickson) and analyzed using FlowJo software (Tree Star Inc.).

Galvani S., Sanson M., Blaho V.A., Swendeman S.L., Obinata H., Conger H., Dahlbäck B., Kono M., Proia R.L., Smith J.D, & Hla T. (2015). HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation. Science signaling, 8(389), ra79.

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Flow Cytometric Analysis of Murine Immune Cells

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Blood cells were washed twice with staining buffer (Biolegend, San Diego, CA, USA) at 800 rcf for 5 min at 4 °C. Afterwards, they were stained with fluorochrome-conjugated antibodies at a concentration of 1 million cells after blocking of FC receptor (CD16/32, Biolegend, San Diego, CA, USA) for 5 minutes. The following antibodies were used: FoxP3-PE, CD3-PE/Cy7, CD25-APC, CD4-PB, Ly6c-PEcy7, CD115-APC and CD11b-PB all from Biolegend (San Diego, CA, USA). The cells were stained for 30 min on ice in the dark. Erythrocytes were lysed with lysing buffer (BD Pharm Lyse^TM, BD Biosciences, Stockholm, Sweden) for 15 min on ice in the dark. Before intracellular staining for FoxP3, cells were washed to remove unbound extracellular antibodies and incubated with Foxp3 Fixation/Permeabilization buffers (eBioscience, San Diego, CA, USA) for 30 min on ice. Cells were blocked again with CD16/32 prior to incubation with intracellular FoxP3 for 30 min. Cells were resuspended in PBS buffer containing 1% BSA and 0.5 mM EDTA. Cells were run in Gallios flow cytometer (Beckman Coulter, High Wycombe, UK) and analyzed with FlowJo software (Tree Star, Inc. Ashland, OR, USA).

Hsiung S., Knutsson A., Vallejo J., Dunér P., Heinonen S.E., Jönsson-Rylander A.C., Bengtsson E., Nilsson J, & Hultgårdh-Nilsson A. (2018). Hyperglycemia does not affect tissue repair responses in shear stress-induced atherosclerotic plaques in ApoE−/− mice. Scientific Reports, 8, 7530.

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Neutrophil Depletion and Hypoxia Effects

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Wild-type C57BL/6 mice were injected intraperitoneally with vehicle control, anti-Ly6G antibody (500mg in 500ul per mouse, eBioscience, UK), clodronate or PBS containing liposomes (500ul per mouse, ClodronateLiposomes.com). Twelve hours later, the mice were challenged with subcutaneous SH1000 and housed in either normoxia (21% O₂) or hypoxia (10% O₂). At 12 hours following the challenge, clinical assessment of sickness was performed by two separate observers and rectal temperature recorded. Mice were then anaesthetised and 200uL of whole blood cell was collected into vials containing 2mM EDTA. Red cells were twice lysed in red cell lysis buffer (Biolegend) and cells were washed in FACS buffer (PBS+2mM EDTA+2%BSA). Cells were stained using Ly6C FITC (Biolegend), Lineage (CD3, CD19 (both Biolegend), Siglec F (BD Biosceinces) PE, CD115 APC (Biolegend), 7-AAD (Biolegend), CD45 AF700 (Biolegend), Ly6G Pacific Blue (Biolegend). Cells were acquired using a BD Fortessa 6 laser flow cytometer.

Thompson A.A., Dickinson R.S., Murphy F., Thomson J.P., Marriott H.M., Tavares A., Willson J., Williams L., Lewis A., Mirchandani A., Dos Santos Coelho P., Doherty C., Ryan E., Watts E., Morton N.M., Forbes S., Stimson R.H., Hameed A.G., Arnold N., Preston J.A., Lawrie A., Finisguerra V., Mazzone M., Sadiku P., Goveia J., Taverna F., Carmeliet P., Foster S.J., Chilvers E.R., Cowburn A.S., Dockrell D.H., Johnson R.S., Meehan R.R., Whyte M.K, & Walmsley S.R. (2017). Hypoxia determines survival outcomes of bacterial infection through HIF-1alpha dependent re-programming of leukocyte metabolism. Science immunology, 2(8), eaal2861.

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Immunophenotyping of Apo-E Mutant Mice

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Splenocytes and blood leukocytes from ApoE-null and ApoE/mdx- mice were stained with flourochrome-conjugated antibodies and were captured and analysed with a CyAn ADP flow cytometer (Beckman Coulter, High Wycombe, UK) using Summit software (Dako, Fort Collins, Colorado, USA). Erythrocytes were removed using red blood cell lysing buffer (Sigma, St. Louis, MO, USA). Fc receptors were blocked before extra- and intracellular staining (FcR; CD16/32). The following antibodies were used; CD3-PE/Cy7, CD4-PB, IL-5-PE, IFNγ-PE, IL-17-APC, Ly6c-PE/Cy7 and CD115-APC (Biolegend, San Diego, CA).

Shami A., Knutsson A., Dunér P., Rauch U., Bengtsson E., Tengryd C., Murugesan V., Durbeej M., Gonçalves I., Nilsson J, & Hultgårdh-Nilsson A. (2015). Dystrophin deficiency reduces atherosclerotic plaque development in ApoE-null mice. Scientific Reports, 5, 13904.

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Quantification of Circulating Monocytes and Lymphocytes

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Circulating monocytes were determined in 10 ml of heparinised whole blood incubated for 30 min at RT with Ly6C-PerCP (BD Pharmingen, Madrid, Spain) and CD115-APC (Biolegend, San Diego, CA, USA). For lymphocytes, 10 ml of heparinised whole blood was incubated for 30 min RT with 5 ml Brilliant Stain Buffer (563794, BD) and with Brilliant violet (BV)-rat anti-mouse CD4 (562891, BD), BV-rat anti-mouse CD8a (563068, BD), PE-hamster anti-mouse CD69 (553237, BD) and APC-hamster antimouse CD3e (553066, BD). Incubation with lysing solution (BD Facs Lysing solution) was done before analysis by flow cytometry (FACSVerse BD Biosciences). To detect functionally-polirised CD4CT lymphocyte, 100 ml of heparinised whole blood was stained with the mouse Th17/Treg phenotyping kit (BD Pharmingen, Madrid, Spain) to detect CD4CFoxp3C and CD4CIL17 cells. Analysis of Ly6C low -and Ly6C hi -subsets were determined in CD115C populations.

Andrés-Blasco I., Herrero-Cervera A., Vinué Á., Martínez-Hervás S., Piqueras L., Sanz M.J., Burks D.J, & González-Navarro H. (2015). Hepatic lipase deficiency produces glucose intolerance, inflammation and hepatic steatosis. The Journal of endocrinology, 227(3).

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Multiparameter Flow Cytometry Immunophenotyping

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Single-cell suspensions were incubated with human Fc receptor-binding inhibitor (Fc Block, eBioscience) before surface staining with anti-human monoclonal antibodies (mAbs). Fluorescence minus one (FMO) controls with isotype controls were used to define positive signals for flow cytometry or cell sorting. Dead cells were excluded with the Zombie Violet Fixable Viability Kit (Biolegend).
For labeling, cells were resuspended in PBS, 2% FCS (1 to 5 x10 7 cells/500 ml) and incubated with the following mAbs: CD45 A700 (Biolegend, clone HI30), CD34 PB (Biolegend, clone 581), CD38 BV510 (BD Bioscience, clone HIT2), CD117 BV605 (Biolegend, clone 104D2), CD45RA PE (BD Bioscience, clone HIT100), CD7 FITC (Beckman Coulter, clone 8H8.1), CD2 PerCPCy5.5 (Biolegend, clone RPA2.10), CD115 APC (Biolegend, clone 9-4D2-1E4), CD116 APC-vio770 (Miltenyi, clone REA211), CD123 BV786 (BD Bioscience, clone 7G3), CD127 PC5 (Biolegend, clone A019D5), ITGB7 PC7 (eBioscience, clone FIB504), CD10 BV650 (BD bioscience, clone HI10a), CD19 BV711 (BD bioscience, clone HIB19), CD24 PE-CF594 (BD bioscience, clone ML5). Flow cytometry analyses and cell sorting were performed with a BD Fortessa Analyzer or a BD FACSAria IIII sorter (BD Biosciences; (purity R 95%).

Alhaj Hussen K., Vu Manh T.P., Guimiot F., Nelson E., Chabaane E., Delord M., Barbier M., Berthault C., Dulphy N., Alberdi A.J., Burlen-Defranoux O., Socié G., Bories J.C., Larghero J., Vanneaux V., Verhoeyen E., Wirth T., Dalod M., Gluckman J.C., Cumano A, & Canque B. (2017). Molecular and Functional Characterization of Lymphoid Progenitor Subsets Reveals a Bipartite Architecture of Human Lymphopoiesis. Immunity, 47(4).

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