For pulse labeling of RNA synthesis, C. uncinata cells were incubated in filtered and autoclaved pond water containing 1 mM 5-ethynyl uridine (EU; Invitrogen) for 30 min directly on Superfrost microscope slide (Fisher). Cells were then fixed in 2% paraformaldehyde solution in phosphate buffer solution (PBS) for 30 min. Fixed cells were then washed in PBS and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. EU labeling was carried out according to the manufacturer’s instructions (Invitrogen; Click-iT RNA labeling kits). The cells were incubated in a 1× working solution of Click-iT reaction solution for 30 min at room temperature. Subsequently, the slides were washed once with Click-iT reaction rinse buffer then once more with PBS. Following this, DNA was counterstained with 0.1 µg/ml 4′,6-diamidino-2-phenyl-indole (DAPI) for 1 min in the dark. Cells were then washed twice with PBS and a drop of SlowFade Gold was added prior to sealing with nail polish.
5 ethynyl uridine
Manufactured by Thermo Fisher Scientific
5-ethynyl uridine is a nucleoside analog that can be used as a labeling reagent for the detection and quantification of newly synthesized RNA in cell culture and in vivo studies. It functions by being incorporated into newly synthesized RNA, allowing for the visualization and analysis of RNA dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost microscope slide, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Click it cell reaction buffer kit, by Thermo Fisher Scientific (1 mentions)
Mnase, by New England Biolabs (1 mentions)
Tetramethylrhodamine dextran, by Thermo Fisher Scientific (1 mentions)
Triptolide, by Merck Group (1 mentions)
Cordycepin, by Fujifilm (1 mentions)
N acetylcysteine, by Fujifilm (1 mentions)
Triptolide, by Adipogen (1 mentions)
5 6 dichloro 1 β d ribofuranosylbenzimidazole drb, by Cayman Chemical (1 mentions)
Ro 3306, by Selleck Chemicals (1 mentions)
Ve 822, by Selleck Chemicals (1 mentions)
Pha 767491, by Merck Group (1 mentions)
Click it eu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
Click it rna imaging kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using 5 ethynyl uridine
1
RNA Synthesis Pulse Labeling in C. uncinata
2
Quantifying RNA Transcription
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Transcription was analyzed by measuring the incorporation of Click‐iT chemistry‐detectable 5‐ethynyl uridine (EU) (C10327; Invitrogen) as described before [43 (link)]. In short, 2 × 103 cells per well were seeded in 384‐well μClear imaging plates. The next day, cells were pre‐treated for 24 h and washed and treatment was pursued in the presence of 1 mM 5‐ethynyl uridine (EU) for 1 h. Following, the cells were fixed with 3.7% formaldehyde supplemented with 1 μg/mL Hoechst 33342 for 1 h and permeabilized with 0.5% Triton X‐100 for 15 min. Alexa Fluor‐488‐coupled azide was then added for 1 h. The intensity of the GFP signal (EU) in the nucleus was measured by microscopy, and the inhibition of transcription was calculated as a fold change in fluorescence intensity as compared to controls.
3
EU Labeling and Immunostaining of XPB-YFP
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XPB-YFP MDF were grown for 24 h on coverslips before being incubated for 30 min with 100 µM of 5-ethynyl uridine (EU from ThermoFisher). After washes with PBS, cells were fixed with 3.7% paraformaldehyde for 15 min at 37 °C, then incubated 2 times for 5 min with PBS and 3% BSA. Cells are then permeabilized for 20 min with PBS 0.5% triton X-100. After 30 min of incubation with PBS 0.5% BSA and 0.15% glycine, cells were in contact for 2 h at room temperature with a primary antibody: RNAP2 (clone 8WG16, 1/100 mouse, Santa Cruz Biotechnology sc56767), HA for XPB staining (1/200, rat, Roche 3F10), TBP (1/200, rabbit, cell signalling 8515S) and p62 (1/100, rabbit, abcam ab204168). After several washes with PBS 0.1% triton X-100, cells were incubated for 1 h with a secondary antibody: Alexa-488 nm donkey anti-rat or goat anti-mouse or goat anti-rabbit (Invitrogen) (dilution 1/400). Cells were washed several times with PBS 0.1% triton X-100 and then incubated for 30 min with Click-iT reaction cocktail containing Alexa Fluor azide 594. After washing, the coverslips were mounted with Vectashield (Vector laboratories).
4
Quantifying RNA Synthesis in Etoposide-Treated Cells
5
RNA Labeling and Uptake in Bladder Cells
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To label MV RNA, UPEC 536 were grown with 100μM 5-ethynyl uridine (5EU; Thermo Fisher Scientific) for 6h at 37°C, shaking at 200rpm, prior to MV isolation. 5637 bladder carcinoma cells were treated with 5EU-labelled membrane vesicles (10μg protein) and stained with CellTracker Red as above. Cells were washed twice with 1% BSA in PBS, fixed and permeabilised as above before being incubated in Click-iT® reaction cocktail (Click-iT Cell Reaction Buffer Kit, Thermo Fisher Scientific) containing 2.5μM azide-containing Alexa Fluor 488 (Thermo Fisher Scientific), according to the manufacturer’s instructions. The nucleus was highlighted with DAPI and visualised as prior.
6
Zscan4 Silencing and DNA Damage Analysis in Mouse Embryos
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The siRNA diced pools against Zscan4 and luciferase (control) were generated using recombinant Giardia lamblia Dicer. Experimental setup was done as previously described (39 (link)). Briefly, 4 μM siRNAs against Zscan4 or luciferase control siRNA were coinjected with dextran-tetramethyl-rhodamine (Invitrogen, 3000 molecular weight 100 μg/ml) in late G1 stage (4 to 5 hpf) zygotes derived from in vitro–fertilized mouse oocytes. After incubation at 37°C in a humidified atmosphere of 5% CO2 and 95% air, Zscan4 or control siRNA–injected 2C embryos at 30 hpf were briefly washed in M2 medium, treated with acidic tyrodes, and fixed in 3.7% paraformaldehyde in PBS at 4°C. After permeabilization with 0.2% Triton X-100 in PBS for 10 min at RT, embryos were blocked overnight at 4°C in 1% BSA and 0.1% Triton X-100 in PBS. For DNase/MNase (micrococcal nuclease) treatment, fixed and permeabilized embryos were incubated with TURBO DNase 1 (2 U/μl, Ambion) and MNase (80 gel units, NEB) for 2 hours at 37°C under mineral oil. Embryos were analyzed for Zscan4 γH2A.X by immunostaining. For triptolide experiments, 2C embryos at 29 hpf were incubated with triptolide (10 μM; Sigma-Aldrich) or dimethyl sulfoxide (DMSO) for 2 hours in the presence of 5-ethynyl-uridine (5 mM; EU, Thermo Fisher Scientific) before fixation at 31 hpf and analyzed for Zscan4, γH2A.X-foci, and EU signals.
7
Small Molecule Inhibitor Protocol
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The ATR inhibitor VE822 and the CDK1 inhibitor (RO-3306) were purchased from Selleck (S7102) and Calbiochem (217699) respectively. Hydroxyurea (HU) was purchased from TCI Chemicals (H0310), Cordycepin and N-Acetyl-cysteine (NAC) were purchased from Wako (017-05131), 5,6-dichloro-1-β-dribofuranosylbenzimidazole (DRB) from Cayman Chemical (10010302), and triptolide from AdipoGen (AG-CN2-0448). 5-Ethynyl Uridine (EU) was purchased from Thermo Fisher Scientific (E10345). CDC7 inhibitor PHA-767491 was purchased from Sigma-Aldrich (PZ0178). Concentrations and conditions used in each assay are listed in Figure legends.
8
Visualizing Newly Synthesized RNA
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Analysis of newly synthesised RNA in cells was achieved by treatment with 2 mM 5-ethynyl uridine (EU) (ThermoFisher) for 20 min before fixation. Replicating cells were visualized following the protocol from Click-iT EU Alexa Fluor 488 Imaging Kit (ThermoFisher).
9
Optogenetic Droplet Formation in HEK293 Cells
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Before serum starvation, the HEK293 cells were transfected with the Cry2 fusion constructs using Lipofectamine 3000 and cultured in DMEM medium with 10% FBS for 24 h. Subsequently, cells were harvested and seeded in eight-chamber slides in DMEM medium without FBS overnight. The medium was replaced by 20% FBS DMEM medium for one hour (for nascent RNA labeling, the 20% FBS medium was mixed with 1 mM 5-ethynyl uridine (Thermo Fisher, cat # C10329)). The cells were exposed under blue light for 10 min to induce optogenetic droplet formation and immediately fixed by 4% formaldehyde in PBS for 20 min at room temperature to be ready for the further staining experiment. We used a Click-it RNA imaging kit (Thermo Fisher, cat # C10329) to stain nascent RNA followed a standard protocol provided by the kit.
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