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Af3239

Manufactured by R&D Systems
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AF3239 is a recombinant mouse Epidermal Growth Factor (EGF) protein. EGF is a potent mitogenic factor that stimulates the growth of various cell types.

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Lab products found in correlation

Ab283655, by Abcam (2 mentions) Nbp1 33634, by Novus Biologicals (2 mentions) Clone mopc 21, by BioXCell (1 mentions) Clone 17f3, by BioXCell (1 mentions) Ab239731, by Abcam (1 mentions) Ab300421, by Abcam (1 mentions) Pannoramic viewer software, by 3DHISTECH (1 mentions) Ab32520, by Abcam (1 mentions) Ab263947, by Abcam (1 mentions) Ab109457, by Abcam (1 mentions) Ab156769, by Abcam (1 mentions) Ab125028, by Abcam (1 mentions) Ab133543, by Abcam (1 mentions) Ab109320, by Abcam (1 mentions) Ab9485, by Abcam (1 mentions) P1511, by Applygen (1 mentions)

3 protocols using af3239

1

Modulating Retinal Vascularization in OIR

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P12 mice pups were anesthetized in 3% isoflurane with oxygen and injected intravitreally with 2 μl of CM, whereas basal αMEM (vehicle) was injected in the contralateral eye as control using a Hamilton syringe equipped with 50-gage glass capillary. 2 μl of mouse recombinant Sema3E Fc Chimera (20 ng/μl; R&D systems) or PBS were administered into vitreous cavity of P12 OIR pups. To assess the effect of blocking IL-17A on the retinal vascularization, 2 μl of 5 μg/μl neutralizing monoclonal IL-17A antibody (Clone 17F3, BioXCell) or of 5 μg/μl mouse IgG1 isotype control monoclonal antibody (Clone MOPC-21, BioXCell) was intravitreally injected. To reverse the benefits of Sema3E induced by MSCs-CM administration during OIR, the eyes were injected at P12 and P14 with 2 μl of blocking Sema3E (5 μg/μl) antibody (AF3239, R&D Systems). To assess the dose-response of MSCs on vasoobliteration, 50,000, 100,000, and 200, 000 cells from passages 3–5 were intravitreally injected in a volume of 2 μl. Retinal vasculature was analyzed in whole-mounts at P17.
Noueihed B., Rivera J.C., Dabouz R., Abram P., Omri S., Lahaie I, & Chemtob S. (2021). Mesenchymal Stromal Cells Promote Retinal Vascular Repair by Modulating Sema3E and IL-17A in a Model of Ischemic Retinopathy. Frontiers in Cell and Developmental Biology, 9, 630645.
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2

Immunofluorescence Analysis of Vascular Markers

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The fixed arteries were embedded in paraffin, and then 4-μm sections were mounted on glass slides. Hematoxylin and eosin (H&E) and immunofluorescence staining were performed following standard protocols [18 (link)]. For immunofluorescence staining, the following primary antibodies were used: PLXND1 (NBP1-33634, Novus), iNOS (ab283655, Abcam), Liver Arginase (ab239731, Abcam), F4/80 (ab300421, Abcam) and SEMA3E (AF3239, R&D Systems). The slides were scanned on a Pannoramic 250 system (3DHISTECH Ltd.), and images were analyzed using the Pannoramic Viewer software (3DHISTECH Ltd.). The percent fluorescent positive area was calculated as the fluorescent signal area divided by the entire intima area and quantified using the FIJI software (version 2.1.0/1.53c, National Institutes of Health, Bethesda, MD, USA).
Zhang S., Zhang Y., Zhang P., Wei Z., Ma M., Wang W., Tong W., Tian F., Hui H., Tian J, & Chen Y. (2023). Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis. Heliyon, 9(6), e17314.
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3

Western Blot Analysis of Macrophage Markers

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Cells were washed twice with cold PBS, collected using cell scrapers and transferred to pre-chilled tubes. Collected cells were centrifuged at 500g for 5 min, and cell pellets were stored at −80 °C. Proteins were extracted from cell pellets using a lysis buffer for western blotting and their concentrations were measured by bicinchoninic acid assay (Applygen Technologies, P1511). Western blotting was performed according to standard procedures [25 (link)]. The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control. Western blots were quantified using the FIJI software. Uncropped Western blot gels are available in the Supplementary Information section.
Zhang S., Zhang Y., Zhang P., Wei Z., Ma M., Wang W., Tong W., Tian F., Hui H., Tian J, & Chen Y. (2023). Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis. Heliyon, 9(6), e17314.
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