C10269

Manufactured by Thermo Fisher Scientific

Sourced in Germany

The C10269 is a laboratory equipment product from Thermo Fisher Scientific. It is designed to perform a specific core function, but without further details or interpretation, a concise and unbiased description cannot be provided. Additional information would be required to describe the product's capabilities accurately and objectively.

Automatically generated - may contain errors

Lab products found in correlation

5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (2 mentions) Scanr analysis software, by Olympus (2 mentions) Ix83 scanr microscope, by Olympus (2 mentions) Biotin alkyne, by Thermo Fisher Scientific (1 mentions) Methionine free dmem, by Thermo Fisher Scientific (1 mentions) E10187, by Thermo Fisher Scientific (1 mentions) Alexa fluor 405 goat anti mouse, by Thermo Fisher Scientific (1 mentions) Alexa fluor 555 goat anti rabbit, by Thermo Fisher Scientific (1 mentions) Alexa fluor 555 goat anti mouse, by Thermo Fisher Scientific (1 mentions) Anti pcna clone pc10, by Santa Cruz Biotechnology (1 mentions)

5 protocols using c10269

EdU Incorporation Assay for Cell Cycle

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10.000 cells were plated per well of a 96 well optical plate. After 24 hours, cells were incubated with 10 μM EdU (Life Technologies, A10044) for 30 minutes, before fixation in 4% PFA. Cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min and washed three times with PBS. Click-iT reaction cocktail containing CuSO₄, L-ascorbic acid and Alexa Fluor 647 fluorescent dye azide in PBS was added to each well and incubated according to the manufacturer’s protocol (Invitrogen C10269). Nuclei were counterstained with DAPI, and cell cycle profiles were made by image acquisition on an automated Olympus IX83 ScanR microscope and Olympus ScanR analysis software.

Albers E., Avram A., Sbroggio M., Fernandez-Capetillo O, & Lopez-Contreras A.J. (2020). Supraphysiological protection from replication stress does not extend mammalian lifespan. Aging (Albany NY), 12(7), 5612-5624.

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EdU Incorporation and Cell Cycle Analysis

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10.000 cells were plated per well of a 96 well optical plate. After 24 hours, cells were incubated with 10 µM EdU (Life Technologies, A10044) for 30 minutes, before fixation in 4% PFA. Cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min and washed three times with PBS. Click-iT reaction cocktail containing CuSO 4 , L-ascorbic acid and Alexa Fluor 647 fluorescent dye azide in PBS was added to each well and incubated according to the manufacturer's protocol (Invitrogen C10269). Nuclei were counterstained with DAPI, and cell cycle profiles were made by image acquisition on an automated Olympus IX83 ScanR microscope and Olympus ScanR analysis software.

Albers E., Avram A., Sbroggiò M., Fernández-Capetillo Ó., & López‐Contreras A.J. (2020). Supraphysiological protection from replication stress does not extend mammalian lifespan.

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Quantifying Newly Synthesized Proteins

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48 hours post-transfection, N2A cells were washed 2X with DPBS and incubated with Methionine-free DMEM (Thermo, 21013024) for 1 hour. After 30 minutes, Anisomycin was added (20 μM) to some wells for the remaining 30 minutes. Afterwards, the methionine analog, AHA (100 μM; Thermo, C10102), was added to cells in Met-free media for 1 hour. The cells were then washed with DPBS. and fixed in 4% paraformaldehyde for 20 minutes at room temperature. The Click-it labeling was performed according to the manufacturer’s recommendations for both western- and fluorescent-based readouts. Fluorescent-based (Thermo, C10269): cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and washed 3X in DPBS. The cells were then permeabilized with 0.25% TritonX-100 for 10 minutes and the click-it assay was performed (4 μM 647-alkyne; Thermo, A10278).
For analysis, images were acquired on a 780 Zeiss confocal microscope and GFP+ cells were outlined and the fluorescence intensity in the 647 channel was measured in Fiji (Schindelin et al., 2012 (link)). Fluorescence intensity was normalized to no AHA controls. Western-based (Thermo, C10276): cells were lysed in 50 mM Tris pH 8, 0.1% SDS + protease inhibitors and then used for the click-it reaction (40 μM biotin-alkyne; Thermo, B10185).

Lennox A.L., Hoye M.L., Jiang R., Johnson-Kerner B.L., Suit L.A., Venkataramanan S., Sheehan C.J., Alsina F.C., Fregeau B., Aldinger K.A., Moey C., Lobach I., Afenjar A., Babovic-Vuksanovic D., Bézieau S., Blackburn P.R., Bunt J., Burglen L., Campeau P.M., Charles P., Chung B.H., Cogné B., Curry C., D’Agostino M.D., Di Donato N., Faivre L., Héron D., Innes A.M., Isidor B., Keren B., Kimball A., Klee E.W., Kuentz P., Küry S., Martin-Coignard D., Mirzaa G., Mignot C., Miyake N., Matsumoto N., Fujita A., Nava C., Nizon M., Rodriguez D., Blok L.S., Thauvin-Robinet C., Thevenon J., Vincent M., Ziegler A., Dobyns W., Richards L.J., Barkovich A.J., Floor S.N., Silver D.L, & Sherr E.H. (2020). Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development. Neuron, 106(3), 404-420.e8.

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In Vivo EdU Labeling of Proliferating Cells

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EdU (E10187, Thermo Fisher Scientific) and thymidine (T9250, Thermo Fisher Scientific) were weighed and reconstituted in DPBS at 2.5 mg/ml and 25 mg/ml, respectively. At E13.5, EdU solution was intraperitoneally (IP) injected into pregnant female mice at 50 mg/kg body weight. After 10 mins, thymidine solution was IP injected at 500 mg/kg to quench EdU incorporation. The injected females were euthanized by CO₂ inhalation followed by cervical dislocation. The embryonic intestines were fixed and sectioned, as described, then subjected to EdU staining after permeabilization. The EdU staining was performed according to the manufacturer’s protocol (C10269, Thermo Fisher Scientific). Briefly, 200 μl of Click-iT reaction cocktail was incubated with each slide for 30 min in the dark. After two washes with 3% BSA in DPBS, Hoechst staining was performed as described in “Immunofluorescence Staining”.

Yang Y., Paivinen P., Xie C., Krup A.L., Makela T.P., Mostov K.E, & Reiter J.F. (2021). Ciliary Hedgehog signaling patterns the digestive system to generate mechanical forces driving elongation. Nature Communications, 12, 7186.

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In Vivo Cell Proliferation Assay in Fish

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BrdU or EdU were dissolved at a concentration of 1 mg/ml in saline solution (0.07% NaCl) containing methylene blue and injected intraperitoneally (5 ul/ 0.1 g body mass) into the fish at precise time points. Fish were over-anesthetized in MS-222 or placed in ice water for euthanasia, decapitated, and the brains dissected and fixed in 4% PFA overnight. Whole mount brains were processed for Click chemistry with Azyde-Alexa-Fluor-647 to detect EdU, following the manufacturer’s protocol (C-10269, Thermo Fisher Scientific, Darmstadt, Germany); see also [68 (link)]. For the subsequent BrdU immunoreaction, brains were treated with HCl 2M at 37°C for 30 min, washed in sodium tetraborate buffer 0.1 M, pH8 and in PBS, and incubated with Mouse-anti BrdU (Phoenix-Flow Systems, San Diego, USA, PRB1-U) 1:800 overnight. Mouse or Rabbit anti-PCNA (clone PC-10, Santa Cruz, Santa Cruz, USA, sc-56; Abcam ab15497) was diluted 1:800 or 1:100, respectively. Following secondary antibodies were incubated for 1 h at room temperature at 1:1,000 concentration: Goat-anti-mouse Alexa-Fluor-555, Goat-anti-mouse Alexa-Fluor-405, Goat-anti-Rabbit-Pacific Blue, and Goat-anti-Rabbit Alexa-Fluor-555 (Thermo Fisher Scientific).

Lupperger V., Marr C, & Chapouton P. (2020). Reoccurring neural stem cell divisions in the adult zebrafish telencephalon are sufficient for the emergence of aggregated spatiotemporal patterns. PLoS Biology, 18(12), e3000708.

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