Cd115 pe

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD115-PE is a fluorophore-conjugated antibody that binds to the CD115 cell surface antigen. It is used for the identification and enumeration of CD115-positive cells in flow cytometry analysis.

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Close spelling variants of this product

CD115-PE-Cy7 (5 citations) Anti‐CD115‐PE (4 citations) Anti-CD115(CSF-1R)-PE (3 citations) PE-labeled anti-mouse CD115 (2 citations) PE anti-mouse CD115 (2 citations) PE-Cy5-conjugated rat monoclonal anti-CD115 (2 citations) CD115 PE conjugated (clone (2 citations)

6 protocols using cd115 pe

Blood Collection and Cell Profiling in Mice

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To obtain samples from the blood, mice were bled via saphenous vein into 10 μL of heparin (25 mg/mL; Sigma-Aldrich, Cat. H3149). 30 μL of heparinized blood was depleted of red blood cells with Ack (Lonza, Cat. 10-548E). Blood cells were blocked with unlabeled anti-CD16/32 mAb followed by the addition of mAbs as indicated in a final volume of 50 uL. Cells were typically labeled with the following panel of monoclonal antibodies: CD19 AF647 (clone: eBio1D3; 1:200; eBioscience), TCRβ APC (clone: H57-597; 1:200; eBioscience), Ly6G Pacific Blue (clone: 1A8; 1:400; Biolegend) CD115 PE (clone: AFS98; 1:100; eBioscience), CD11b PerCp-Cy5.5 (clone: M1/70; 1:600; eBioscience), Ly6C FITC (clone: HK1.4; 1:400; Biolegend) and CD45.2 Alexa Flour 700 (clone: 104; 1:200; eBioscience). Samples were incubated at 4° C for 30 minutes for surface stains, then washed and fixed in 4% paraformalydehyde. Blood cell numbers were quantified using polystyrene microspheres (Polysciences, Cat. 18328). The same animal was tracked longitudinally across all time points for determination of cell numbers in the blood. Data for all time points was acquired on the same day using the same bead lot on an LSRII (BD Biosciences) and analyzed using FlowJo (TreeStar). Live cell were analyzed by using FSC and SSC. BM pDC were isolated and sorted as described previously [25 (link)].

Buechler M.B., Gessay G.M., Srivastava S., Campbell D.J, & Hamerman J.A. (2015). Hematopoietic and Non-Hematopoietic Cells Promote Type I Interferon- and Toll-like Receptor 7-dependent Monocytosis During Low-dose Lymphocytic Choriomeningitis Virus Infection. European journal of immunology, 45(11), 3064-3072.

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Quantification of Monocyte and Neutrophil Subsets

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Peripheral blood was collected by submandibular vein puncture into heparin-containing tubes. Red blood cells were removed from flow cytometry preparations by treatment with ACK lysing buffer (Gibco, no. A10492). The remaining white blood cells were resuspended in Hanks’ balanced salt solution (0.1% BSA, 5 mM EDTA). Blood cells were then incubated with FLICA 660-YVAD-FMK (ImmunoChemistry Technologies, no. 9122) at 1:150 dilution in RPMI-1640 medium plus 5% FBS at 37°C for 1 h, followed by staining for monocytes and neutrophils using a cocktail of antibodies against CD45-APC (BD Pharmingen, no. 559864), Ly6-C/G-PerCP-Cy5.5 (BioLegend, no. 108427), and CD115-PE (eBioscience, no. 12-1152). Monocytes were identified as CD45⁺CD115⁺ and further separated into Ly6C^high and Ly6C^low subsets, and neutrophils were identified as CD45⁺CD115⁻Ly6G⁺. Data were acquired on a BD FACS Canto II instrument (BD Biosciences, Becton, Dickinson and Co., Franklin Lakes, NJ) and analyzed using FACSDiva software (version 6.1.3, BD Biosciences).

Shen L., Yang Y., Ou T., Key C.C., Tong S.H., Sequeira R.C., Nelson J.M., Nie Y., Wang Z., Boudyguina E., Shewale S.V, & Zhu X. (2017). Dietary PUFAs attenuate NLRP3 inflammasome activation via enhancing macrophage autophagy. Journal of Lipid Research, 58(9), 1808-1821.

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Comprehensive Phenotypic Profiling of Immune Cells

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The following reagents were procured as described: CD11c-FITC, CD11c-PE, CD11b-APC, I MHC class II (I-Ad/I-Ed)-FITC, CD40-V421 (BD Bioscience, USA), CD80-FITC, CD86-PE, CD206-PE, CD14-PE, CD115-PE, F4/80-APC, CD103-PE, CD8a-V430, CD197-PE, B220-PE, CD207-PE, DEC-205-PE, CD124-PE, sigH-PE (eBioscience, USA). Blocking antibodies against TRAIL, FasL, TNFR1 (Biolegend, USA), ultrapure mouse rIFNβ, rTNFα (Biolegend, USA). DAPI, Cell Trace Violet (Invitrogen, USA), L-NMMA, β-Mercaptoethanol (Sigma, USA), FeTPPS (Calbiochem, USA), Bx795 (Invitrogen, USA). LPS 055:B5 (TLR tested, Invivogen, USA). LTA-BS, Pam2CSK4, Pam3CSK4, Poly(I:C) HMW, Flagellin-PA Ultrapure, Imiquimod, ODN CpG 1826 (Invivogen, USA). Pharmaceutical grade Immunomax® (IMM) has been purchased from Immapharma Ltd (Russia).

Bagaev A., Pichugin A., Nelson E.L., Agadjanyan M.G., Ghochikyan A, & Ataullakhanov R.I. (2018). Anti-cancer mechanisms in two murine bone marrow derived-DC subsets activated with Toll-like receptor 4 agonists. Journal of immunology (Baltimore, Md. : 1950), 200(8), 2656-2669.

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Exosomal Nef Modulates Monocyte Phenotypes in Mice

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C57BL/6 mice were purchased from Jackson Laboratories and colonies were maintained at AMREP. Mice were housed in a normal light and dark cycle and had ad libitum access to food (normal chow) and water. C57BL/6 mice were administered either exNef (2μg, I.V.) or control (exGFP) exosomes (2μg, I.V.), 3 times a week, for a period of 2 weeks. At the end of the experiment mice were euthanized and blood was collected via cardiac puncture into EDTA tubes, which were immediately placed on ice. Liver and spleen were excised and peritoneal macrophages were collected after washing peritoneal cavity with PBS.
For flow cytometry analysis, red blood cells were lysed (BD pharm Lyse; BD Biosciences), and white blood cells were centrifuged, washed, and resuspended in HBSS (0.1% BSA w/v, 5 mM EDTA). Cells were stained with a cocktail of antibodies against CD45-PB, Ly6-C/G-PerCP-Cy5.5 (BD Biosciences), CD115-PE (eBioscience) and CTxB-FITC. Monocytes were identified as CD45^hiCD115^hi and further subdivided into Ly6-C^hi and Ly6-C^lo. CTB binding was measured as mean fluorescence intensity (MFI). Samples were run on the LSR Fortessa, and analysed using FlowJo. Plasma total cholesterol, HDL cholesterol and triglyceride content were measured by colorimetric assays from Wako (Japan) according to manufacturer’s instructions.

Mukhamedova N., Hoang A., Dragoljevic D., Dubrovsky L., Pushkarsky T., Low H., Ditiatkovski M., Fu Y., Ohkawa R., Meikle P.J., Horvath A., Brichacek B., Miller Y.I., Murphy A., Bukrinsky M, & Sviridov D. (2019). Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells. PLoS Pathogens, 15(7), e1007907.

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Immune Cell Profiling in Murine BMDMs

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After removing the red blood cells using ACK lysing buffer (Gibco), peripheral white blood cells were stained with FLICA 660-YVAD-FMK (1:150 dilution, ImmunoChemistry Technologies), CD115-PE (eBioscience, no. 12-1152), Gr1(Ly6C/G)-PerCP-Cy5.5 (BioLegend, no. 108427), and CD45-APC (BD Pharmingen, no. 559864).
Data were acquired on a BD FACS Canto II instrument (BD Biosciences) and analyzed using FlowJo analytical software (version 7.6.5, TreeStar) . BMDMs were stained with 7-AAD (Invitrogen, cat# V35123) or PI (Invitrogen, Cat# P3566) according to the manufacturer's instructions. Data were acquired on a BD FACSCalibur and analyzed using FlowJo analytical software (version 7.6.5, TreeStar).

Meyers A.K., Wang Z., Han W., Zhao Q., Zabalawi M., Duan L., Liu J., Zhang Q., Manne R., Lorenzo F.R., Quinn M., Song Q., Fan D., Lin H., Furdui C.M., Locasale J.W., McCall C.E., & Zhu X. (2021). Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation.

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Immune Cell Profiling in Irradiated Mice

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Female C57BL/6J mice aged 12 weeks were purchased from Charles River Laboratories (Saint-Germain-Nuelles, France). The mice were immobilised through anaesthesia (2% isoflurane) and locally irradiated at the thorax using a Varian Tube NDI 226 (X-ray machine; 250 Kev, tube current: 15 mA, beam filter: 0.2 mm Cu), with a dose rate of 1.08 Gy•min -1 . A single dose of 16 Gy was locally administered to the whole thorax.
Flow cytometry and fluorescence-activated cell sorting F4/80-FITC (eBioscience, Paris, France), Gr1-PE (eBioscience), CD11c-PE-Cy7 (Biolegend, Ozyme, Montigny-le-Bretonneux, France), and CD11b-APC-Cy7 (Biolegend) were used to identify neutrophils (Gr1 high (Ly6G + Ly6C -)), monocytes (Gr1 + (Ly6G -Ly6C + )), AMs (CD11c + CD11b -F4/80 + ) [14] and IMs (Gr1 -CD11b + CD11c -/+ F4/80 + ) [15] . Gr1 -mononuclear phagocytes (MPs) were identified as Gr1 - CD11b + CD11c + F4/80 -. Icam1-Pacific blue (Biolegend) was used to identify M1-polarised macrophages and CD206-PerCpCy5.5 (Biolegend), to identify M2-polarised macrophages. CD115-PE (eBioscience) was used to examine CSF1R expression in AMs and IMs. The samples were analysed using FlowJo 10.0.7 (FlowJo, Ashland, OR, USA). IMs and AMs were sorted using fluorescence-activated cell sorting and transferred in the culture medium.

Meziani L., Mondini M., Petit B., Boissonnas A., Thomas de Montpreville V., Mercier O., Vozenin M.C, & Deutsch E. (2018). CSF1R inhibition prevents radiation pulmonary fibrosis by depletion of interstitial macrophages. The European respiratory journal, 51(3).

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