Anti nrp1

Manufactured by R&D Systems

Anti-NRP1 is a recombinant protein that binds to the Neuropilin-1 (NRP1) receptor. NRP1 is a cell surface receptor involved in various cellular processes. Anti-NRP1 can be used in research applications to study the role and function of NRP1.

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Lab products found in correlation

Zombie aqua fixable viability dye, by BioLegend (1 mentions) Anti phospho zap 70, by Cell Signaling Technology (1 mentions) Lsrfortessa x 20, by BD (1 mentions) Ack lysis buffer, by BioLegend (1 mentions) Gentlemacs dissociator, by Miltenyi Biotec (1 mentions) Rabbit anti tbr1, by Abcam (1 mentions) Anti brdu, by Abcam (1 mentions) Anti nrp2, by R&D Systems (1 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Rabbit anti tbr2, by Abcam (1 mentions) Anti gfp, by Takara Bio (1 mentions) Goat anti nrp1, by R&D Systems (1 mentions) Control goat igg, by R&D Systems (1 mentions) Vegf165, by BioLegend (1 mentions)

4 protocols using anti nrp1

Single-cell analysis of tumor-infiltrating immune cells

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Fresh tumor tissue was dissociated at 37 °C for 1 h in a mixture of RPMI, DNaseI, Collagenase Type IV and Dispase using “gentle MACS dissociator” (Miltenyi Biotec). Cells were filtered and washed in 1X PBS −/− buffer and resuspended in 1X ACK lysis buffer (BioLegend, CA) in the dark for 10 min at Room Temperature before dilution in 1X PBS^−/− and washed in FACS buffer (1XPBS, 2% FCS) at 4 °C. Flow cytometry staining was performed on 500,000 cells for membrane phenotype using the following monoclonal antibodies: anti-CD45, CD3, CD4, CD8, PD1 (Beckton Dickinson); anti-NRP1 (R&D Systems) anti-phosphoZAP70 (Cell Signaling Technology). All antibodies were used at concentrations recommended by the manufacturer. Cells were stained for viability using the Zombie Aqua Fixable Viability dye (BioLegend) according to the manufacturer’s protocol. The samples were acquired on the BD LSR Fortessa X-20 (Beckton Dickinson) and analysed using FlowJo v10 software (Tresstar Inc.).

Rossignol J., Belaid Z., Fouquet G., Guillem F., Rignault R., Milpied P., Renand A., Coman T., D’Aveni M., Dussiot M., Colin E., Levy J., Carvalho C., Goudin N., Cagnard N., Côté F., Babdor J., Bhukhai K., Polivka L., Bigorgne A.E., Halse H., Marabelle A., Mouraud S., Lepelletier Y., Maciel T.T., Rubio M.T., Heron D., Robert C., Girault I., Lebeherec D., Scoazec J.Y., Moura I., Condon L., Weimershaus M., Pages F., Davoust J., Gross D, & Hermine O. (2022). Neuropilin-1 cooperates with PD-1 in CD8+ T cells predicting outcomes in melanoma patients treated with anti-PD1. iScience, 25(6), 104353.

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Immunohistochemistry of Neural Markers

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Immunohistochemistry was performed according to the published procedure12 (link) using the following antibodies: anti-Nrp1 (1:500; R&D systems), anti-Nrp2 (1:500; R&D systems), anti-GFP (1:1,000; Clontech), anti-β-galactosidase (1:1,000; ICN Biochemicals), anti-OCAM (1:500; R&D systems), rabbit anti-Tbr1 (1:5,000; Abcam), rabbit anti-Tbr2 (1:5,000; Abcam), anti-BrdU (1:200; Abcam) and rabbit anti-Tbx21 (1:10,000; provided by Dr Yoshihara at RIKEN). Antibodies against Pcdh21 were generated by immunizing guinea pigs with KLH-conjugated synthetic peptides as described previously11 (link). Alexa fluor-conjugated secondary antibodies (Invitrogen) were used at 1:200 dilution.

Inokuchi K., Imamura F., Takeuchi H., Kim R., Okuno H., Nishizumi H., Bito H., Kikusui T, & Sakano H. (2017). Nrp2 is sufficient to instruct circuit formation of mitral-cells to mediate odour-induced attractive social responses. Nature Communications, 8, 15977.

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Sublethal and Lethal Dose TBI Therapy

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For sublethal TBI, adult C57BL/6J mice were irradiated with 500 cGy TBI using a Cesium-137 irradiator. Mice were dosed via intravenous injection (IV) every other day for up to 10 days with 10 µg goat anti-NRP1 (R&D Systems, Minneapolis, MN), or goat IgG control (R&D Systems, Minneapolis, MN), beginning at +24 h of post-TBI.
For lethal dose TBI, adult C57BL/6J mice were irradiated with 800 cGy TBI, which is lethal at our institution for approximately 50% of C57BL/6J mice by day +30 (LD50/30). Mice were dosed IV with either 10 µg goat anti-NRP1 (R&D Systems, Minneapolis, MN) or goat IgG control (R&D Systems, Minneapolis, MN), beginning at +24 h of post-TBI and every other day through day +21.
For evaluation of chemotherapy effects, adult C57BL/6J mice were treated with 5 fluorouracil (5FU), 250 mg/kg IV × 1, and then administered 10 μg anti-NRP1 or goat IgG control every other day IV through day +10.

Termini C.M., Pang A., Fang T., Roos M., Chang V.Y., Zhang Y., Setiawan N.J., Signaevskaia L., Li M., Kim M.M., Tabibi O., Lin P.K., Sasine J.P., Chatterjee A., Murali R., Himburg H.A, & Chute J.P. (2021). Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution. Nature Communications, 12, 6990.

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Monocyte Migration Assay with IFN-β and VEGF

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Cells were pretreated overnight with or without 10ng/ml IFN-β. The following day, cells were washed and resuspended in 5×10⁵/200ul. 200μl of cells were added to an 8-micron pore 24-well transwell plate (Corning). At the bottom of the well, 800μl of regular culture media or 50ng/ml VEGF-165 supplemented media was plated (BioLegend). The assay sat overnight in a CO₂ incubator at 37°C. The next day, transwells were removed and cells were collected at the bottom and counted. Each treatment (PBS control, IFN-β, isotype control, or anti-NRP-1) underwent the assay for migration towards media-alone or VEGF-supplemented media. Fold change is the calculation of the number of cells that migrated towards VEGF divided by the number of cells that migrated in the absence of VEGF. For anti-NRP-1 blocking experiments, overnight rested monocytes were pre-treated with anti-NRP-1 (R&D Systems) or the respective isotype control for 45 minutes on ice and then washed in PBS prior to addition to the transwell.

Nikolai-Yogerst A., White P, & Iwashima M. (2021). IFN-β reduces NRP-1 expression on human cord blood monocytes and inhibits VEGF-induced chemotaxis. Cytokine, 143, 155519.

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