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Af2215

Manufactured by R&D Systems
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Sourced in France

AF2215 is a laboratory reagent manufactured by R&D Systems. It is a purified recombinant protein that functions as a cytokine. The core function of this product is to facilitate the study of cellular processes and interactions involving the specified protein.

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Lab products found in correlation

Ab2074, by R&D Systems (1 mentions) Metavue analyzing software, by Molecular Devices (1 mentions) Alexa fluor 488 igg, by Thermo Fisher Scientific (1 mentions) Af467, by R&D Systems (1 mentions) Af3038, by R&D Systems (1 mentions) Dmr fluorescence microscope, by Leica (1 mentions) Operetta high content imaging system, by PerkinElmer (1 mentions) Harmony high content imaging and analysis software, by PerkinElmer (1 mentions) 96 well plate, by Greiner (1 mentions) Triple express, by Thermo Fisher Scientific (1 mentions) Facsdiva software 8, by BD (1 mentions) Lsrfortessa cell analyzer, by BD (1 mentions) Fortessa, by BD (1 mentions)

4 protocols using af2215

1

Immunofluorescence Staining of Endothelial Cells

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Cells were fixed in 4% paraformaldehyde/PBS for 10 minutes at room temperature and rinsed with PBS 1X. For intracellular staining, cells were permeabilized with 0.1% Triton X100/PBS for 10 minutes at room temperature. Cells were incubated over night at +4°C with antibodies anti-CD144 (1∶200, Beckman Coulter, IM1597), anti-CD31 (1∶20, BD Pharmingen, 550389), anti-ZO1 (1∶200, BD Biosciences, 610966), anti-CL3 (1∶100, Abcam, C0144), anti-CL5 (1∶100, Invitrogen, 34–1600), anti-OCCL (1∶100, Invitrogen, 33–1500), anti-EFNB2 (1∶10, R&D Systems, AF467), anti-NRP1(1∶20, R&D Systems, AF3870), anti-HEY2 (1∶50, Santa Cruz Biotechnology, sc-28747), anti-ANGPT2 (1∶20, R&D Systems, AF623), anti-CXCR4 (1∶100, Abcam, ab2074), anti-COUP-TFII (1∶100, R&D systems, PP-H7147-10), anti-EPHB4 (1∶10, R&D systems, AF3038) or anti-NRP2 (1∶40, R&D Systems, AF2215) antibodies diluted in 3% BSA/PBS and, after 5 PBS 1X washes, labeled with secondary antibodies Alexa Fluor 488 IgG (Invitrogen, Cergy-Pontoise, France). Cells were then stained with 2 µg/mL of 40,6-diamidino-2-phenylindole (DAPI) and examined with a DMR fluorescence microscope (Leica, Rueil Malmaison, France) equipped with a CoolSnap HQ2 camera (Photometrics, Tucson, AZ) controlled by MetaVue® Analyzing Software (Molecular Devices LLC, Sunnyvale, CA).
Ponio J.B., El-Ayoubi F., Glacial F., Ganeshamoorthy K., Driancourt C., Godet M., Perrière N., Guillevic O., Couraud P.O, & Uzan G. (2014). Instruction of Circulating Endothelial Progenitors In Vitro towards Specialized Blood-Brain Barrier and Arterial Phenotypes. PLoS ONE, 9(1), e84179.
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2

Analyzing Arenavirus Infection Inhibition by NRP2 Antibody

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HUVECs, seeded in 96-well plates (Greiner BioOne), were placed on ice for 15min prior to treating with serial dilutions of chilled polyclonal NRP2 antibody (AF2215, R&D Systems) or media alone. Following 30min incubation on ice, cells were exposed to LUJV Zambia R4356 at a MOI of 1 for 1 hour at 37°C, 5% CO2 in the presence of NRP2 antibody or media alone and incubated at 37°C, 5% CO2 for 1 hour. Cells were then washed and incubated with growth medium at 37°C for 36 hours before fixing with 10% formalin. Cells were then permeabilized with 0.02% Triton-X and incubated sequentially with arenavirus NP-specific polyclonal guinea pig serum, then Alexa Flour488-conjugated secondary antibody. Cells were imaged using the Operetta high-content imaging system (PerkinElmer, Waltham, MA) and Harmony high-content imaging and analysis software (PerkinElmer).
Raaben M., Jae L.T., Herbert A.S., Kuehne A.I., Stubbs S.H., Chou Y.Y., Blomen V.A., Kirchhausen T., Dye J.M., Brummelkamp T.R, & Whelan S.P. (2017). NRP2 and CD63 are host factors for Lujo virus cell entry. Cell host & microbe, 22(5), 688-696.e5.
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3

Quantifying CD63 and NRP2 Expression

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HEK293T cells (transduced with the different expression cassettes described in Fig.3C) or HUVEC cells (transduced with a lentivirus expressing a guide RNA targeting CD63) were expanded in 10cm dishes, harvested with TripLE Express (Thermo Fisher Scientific) and stained with an anti-CD63 antibody (clone NKI-C3) together with a goat isotype control antibody or with an anti-NRP2 antibody (AF2215, R&D Systems) in PBS supplemented with 2% FCS. For the polyclonal HUVECs, two cell populations were gated from the living cell pool (i.e. CD63+ and CD63−) and were analyzed for NRP2 cell surface expression using a flow cytometry (LSR Fortessa cell analyzer, BD Biosciences) and FACSDiva Software 8.0.1 (BD Biosciences). 20,000 events were measured per experimental condition.
Raaben M., Jae L.T., Herbert A.S., Kuehne A.I., Stubbs S.H., Chou Y.Y., Blomen V.A., Kirchhausen T., Dye J.M., Brummelkamp T.R, & Whelan S.P. (2017). NRP2 and CD63 are host factors for Lujo virus cell entry. Cell host & microbe, 22(5), 688-696.e5.
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4

Flow Cytometry Staining of HCMV Receptors

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Flow cytometry staining procedures were the same as in our previous work carried out to identify HCMV receptors (5 (link)). For cell surface staining, adherent cells (ARPE-19, HUVEC, and HAP-1) were gently detached with a cell scraper, washed twice with PBS + 2% FBS, and incubated in PBS + 0.5% bovine serum albumin (BSA) + 2 mM EDTA with specific anti-NRP2 and anti-CD46 antibodies (AF2215 and AF2005, R&D Systems), anti-THBD antibody (clone 141C01, Abcam), or, as isotype control, normal sheep immunoglobulin G (IgG) affinity pure (R&D Systems) at 2 μg/ml for 30 min on ice. After two washes, cells were incubated with anti-mouse IgG (H+L) Alexa Fluor 594–conjugated (Thermo Fisher Scientific) secondary antibodies at 2 mg/ml for 30 min on ice, washed twice, and acquired with a FACS (fluorescence-activated cell sorting) Fortessa (BD Biosciences) flow cytometer. Analysis was performed with FlowJo software (TreeStar).
Kschonsak M., Johnson M.C., Schelling R., Green E.M., Rougé L., Ho H., Patel N., Kilic C., Kraft E., Arthur C.P., Rohou A.L., Comps-Agrar L., Martinez-Martin N., Perez L., Payandeh J, & Ciferri C. (2022). Structural basis for HCMV Pentamer receptor recognition and antibody neutralization. Science Advances, 8(10), eabm2536.
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