5 ethynyl 2 deoxyuridine edu

Manufactured by Santa Cruz Biotechnology

5-Ethynyl-2'-deoxyuridine (EdU) is a thymidine analog that can be used to detect and quantify cellular DNA synthesis. EdU is incorporated into DNA during active DNA replication and can be detected using a copper-catalyzed click reaction with an azide-containing fluorescent dye.

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3 protocols using 5 ethynyl 2 deoxyuridine edu

Modulation of Cellular Pathways

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Ganciclovir (GcV, Sigma-Aldrich #G2536), was used at concentration ranging from 1–100mM. Tunicamycin (#11445, Cayman chemicals) was used at 5μg/ml. Sodium 4-Phenylbutyrate (4-PBA, #11323, Cayman chemicals) was used at 5mM. 5-Ethynyl-2’-deoxyuridine (EdU, #sc-284628, Santa Cruz biotechnology) was used at 10mM.

Pommier A., Anaparthy N., Memos N., Kelley Z.L., Gouronnec A., Yan R., Auffray C., Albrengues J., Egeblad M., Iacobuzio-Donahue C.A., Lyons S.K, & Fearon D.T. (2018). Unresolved endoplasmic reticulum stress engenders immune-resistant, latent pancreatic cancer metastases. Science (New York, N.Y.), 360(6394), eaao4908.

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Microglial Depletion and Lineage Mapping

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For acute microglial ablation, mice were administered CSF1R antagonist PLX5622 orally via PLX diet (1200 mg/kg PLX5622, Plexxikon Inc, Berkeley, USA). Control diet (CD) with the same base formula was used as control. For lineage mapping, tamoxifen (Sigma-Aldrich, T5648) was dissolved in corn oil and administered to the CX3CR1-CreERT2/Rosa26-stop-DsRed mice by intraperitoneal (I.P.) injection for 10 days at a daily dose of 2 mg. To label proliferative cells, a solution containing 20 mg/mL 5-Ethynyl-2’-deoxyuridine (EdU) (Santa Cruz, sc-284628) was prepared fresh in sterile PBS and given to mice via I.P. injection at a dose of 80 mg/kg per animal. To maximize EdU labeling, two injections, separated by 7 hr, were given on the same day (Repopulation day 3). To detect EdU-labeled cells in brain sections, Click-iT EdU imaging kits (ThermoFisher Scientific, C10337) were used following the manufacturer’s instructions before the immunofluorescence staining procedures.

Zhan L., Fan L., Kodama L., Sohn P.D., Wong M.Y., Mousa G.A., Zhou Y., Li Y, & Gan L. (2020). A MAC2-positive progenitor-like microglial population is resistant to CSF1R inhibition in adult mouse brain. eLife, 9, e51796.

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Sorting and Analyzing Cancer Cells and CAFs

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For sorting of cancer cells and CAFs, KPC tumors were processed as previously described (19 (link)). Cells were stained with anti-mouse CD45-AlexaFluor 647 (103124, BioLegend), CD326 (Ep-CAM)-AlexaFluor 488 (118212, BioLegend), CD31-AlexaFluor 647 (102416, Biolegend), CD140a (PDGFRα)-PE (135905, BioLegend), PDPN-APC/Cy7 (127418, Biolegend) and DAPI for 15 min. Cells were sorted on the FACSAria cell sorter (BD) for DAPI/CD45/CD31- EpCAM+ and DAPI/CD45/CD31/EpCAM- PDPN+ cell populations. For flow cytometry analysis of EdU-treated KPC tumors, KPC mice were administered 300 μg 5-Ethynyl-2-deoxyuridine (EdU) (61135-33-9, Santa Cruz) formulated in sterile saline twice a day for 3 days via intraperitoneal injection. EdU was detected using Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (C10634, Thermo Fisher Scientific). For flow cytometric analysis of IL-1R1 and myCAF/iCAF populations, antibodies employed were: anti-mouse CD31-PE/Cy7 (102418, Biolegend), CD45-PerCP/Cy5.5 (103132, Biolegend), CD326 (Ep-CAM)-AlexaFluor 488, PDPN-APC/Cy7, CD140a (PDGFRα)-PE, Ly6C-APC (128015, Biolegend), biotinylated CD121a (IL-1R, Type I/p80) (113503, Biolegend) and APC Streptavidin (405207, Biolegend).

Biffi G., Oni T.E., Spielman B., Hao Y., Elyada E., Park Y., Preall J, & Tuveson D.A. (2018). IL-1-induced JAK/STAT signaling is antagonized by TGF-β to shape CAF heterogeneity in pancreatic ductal adenocarcinoma.. Cancer discovery, 9(2), 282-301.

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