Click it reaction

Manufactured by Thermo Fisher Scientific

The Click-iT reaction is a method for the detection and analysis of biomolecules, such as proteins and nucleic acids, through the use of click chemistry. It enables the labeling and visualization of these biomolecules in cells or cell-free systems.

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Click-iT reaction cocktail (53 citations) Click-it cell reaction kit (12 citations) Click-iT Cell Reaction Buffer (5 citations) Click-iT® Protein Reaction Buffer (4 citations) Click-iT reaction buffer (4 citations) Click-iT EdU reaction additive (3 citations) Click-IT reaction buffer wash solution (2 citations) Click-it 488 reaction solution (2 citations) Click-iT® reaction mixture (2 citations) Click-iT cell reaction cocktail (2 citations)

28 protocols using click it reaction

EdU Labeling and Flow Cytometry

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After incubation with 5-ethynyl-2 0 -deoxyuridine (100 mM, EdU) (Life Technologies) for 15 min, cells were subjected to a Click-iT reaction for labeling with Alexa Fluor 647. The reaction was performed according to our original procedure (Sakaue-Sawano et al., 2013) . Cells were fixed with 4% paraformaldehyde (PFA) for 10 min on ice, and then were treated with azide-conjugated Alexa Fluor 647 by the Click-iT reaction (Life Technologies) with a slight modification. As 4 mM copper ion for the conjugation between EdU and Alexa dyes in the Click-iT reaction tended to quench fluorescent proteins, we attempted to lower the copper ion concentration. We found that 1.3 mM copper ion was sufficient for the reaction while effectively avoiding the quenching reaction. Finally, the cell samples were stained with 3 mM DAPI and analyzed using a FACSAria II (BD Biosciences) (Table S1).

Sakaue-Sawano A., Yo M., Komatsu N., Hiratsuka T., Kogure T., Hoshida T., Goshima N., Matsuda M., Miyoshi H, & Miyawaki A. (2017). Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle. Molecular cell, 68(3).

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Quantification of DNA Replication

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Following a 24–48 h transfection with SAMD9/L 293 T cells were treated with 10uM EdU for 2 h. Following the EdU incubation, 293 T cells were harvested and fixed with 4% paraformaldehyde. After fixing, the Click-It® reaction (Invitrogen, C10635) was performed according to the manufacturer’s protocol. Following the Click-It® reaction, total DNA was labeled with FxCycle (Invitrogen, F10347), and analyzed by flow cytometry.

Schwartz J.R., Ma J., Lamprecht T., Walsh M., Wang S., Bryant V., Song G., Wu G., Easton J., Kesserwan C., Nichols K.E., Mullighan C.G., Ribeiro R.C, & Klco J.M. (2017). The genomic landscape of pediatric myelodysplastic syndromes. Nature Communications, 8, 1557.

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Measuring Nascent Protein Synthesis in SARS-CoV-2

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To measure nascent protein synthesis, HeLa cells were plated into 6-well plates and transfected with 1 μg SARS-CoV-2 NSP1 WT, SARS-CoV-2 NSP1 mutant plasmids or SARS-CoV-2 NSP9 the following day. Twenty-four hours post transfection, the existing media was depleted and replenished with methionine-free DMEM (Gibco) supplemented with L-Cysteine (Alfa Aesar) for one hour. For multiplexes with immunofluorescence, cells were incubated with 100 μM homopropargylglycine (HPG) for 2 hours. Cells were washed once with PBS and then fixed with 4% paraformaldehyde. The Click-iT reaction (Thermo Fisher Scientific) was performed according to the manufacturer’s protocol and immunofluorescence performed as described above. For blots, following methionine depletion, cells were incubated with 50 μM of L-azidohomoalanine (AHA) for one hour. Cells were washed three times with PBS, harvested by centrifugation, and lysed with AHA lysis buffer (1% SDS in 50 mM Tris-HCL, pH 8.0) supplemented with protease inhibitor and 1 U/ml Benzonase Nuclease (Sigma). Protein concentration was determined by Bradford reagent. The Click-iT reaction was performed according to the manufacturer’s protocol with 40–50 μg of total protein. Lysates were run on SDS-PAGE gel as described above.

Vazquez C., Swanson S.E., Negatu S.G., Dittmar M., Miller J., Ramage H.R., Cherry S, & Jurado K.A. (2021). SARS-CoV-2 viral proteins NSP1 and NSP13 inhibit interferon activation through distinct mechanisms. PLoS ONE, 16(6), e0253089.

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Detection of DNA-Pt Lesions in Cells

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For DNA–Pt lesion detection, cells were treated with APPO (final [APPO] = 100 μM) and collected at different time points. Then, viable cells were sorted based on their ALDH enzymatic activity using ALDEFLUOR assay. Sorted cells were washed with PBS and preextracted with CSK buffer (10 mM Pipes, pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, and 0.7% Triton X-100) twice for 3 mins. Then, cells were washed with PBS, cytospun and fixed with 4% PFA for 10 min, and permeabilized with Triton X-100 0,1% for 5 min. APPO labeling was done with Click-it reaction (C10269, ThermoFisher Scientific) and alkyne AlexaFluor 488 (A10267, ThermoFisher Scientific). DNA was counterstained with DAPI. For each condition, immunofluorescence scoring was done on 100 cells in three independent experiments.

Azzoni V., Wicinski J., Macario M., Castagné M., Finetti P., Ambrosova K., Rouault C.D., Sergé A., Farina A., Agavnian E., Coslet S., Josselin E., Guille A., Adelaide J., Zacharioudakis E., Castellano R., Bertucci F., Birnbaum D., Rodriguez R., Charafe-Jauffret E, & Ginestier C. (2022). BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells. Cell Death & Disease, 13(2), 96.

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Assessing Cellular Proliferation in GC Cells

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For CCK-8 assays, 5 × 10³ GC cells with indicated transfections were seeded and maintained for 1, 3, 5 or 7 days in 96-well plates. Culture medium was replaced with 100 µL of fresh medium, and CCK-8 (10 µL, Sigma) was added. Subsequently, cells were incubated for 5 hours, and OD 450 was recorded. For EdU incorporation, GC cells were seeded and cultured to 70% confluency. EdU was supplemented added into culture medium at 10 µM, and cells were incubated for 18 h. Then, cells were fixed and permeabilized. The reaction cocktail was prepared, and the Click-iT reaction was performed following the manual (ThermoFisher Scientific). Finally, DAPI was used for nuclear staining, and cells were imaged with an EVOS fluorescence microscope (ThermoFishr Scientific). For colony formation assays, 1 × 10³ GC cells with indicated transfections were seeded and cultured for 14 days in 6-well plates. Subsequently, cell colonies were fixed, stained in 0.1% crystal violet solution (Selleck, Houston, TX, USA) and imaged with the Olympus BX51 microscope (Tokyo, Japan).

Luo M., Deng X., Chen Z, & Hu Y. (2023). Circular RNA circPOFUT1 enhances malignant phenotypes and autophagy-associated chemoresistance via sequestrating miR-488-3p to activate the PLAG1-ATG12 axis in gastric cancer. Cell Death & Disease, 14(1), 10.

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Cell Proliferation Assay with OPP

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protocol was based on a previously described assay (Signer et al., 2014 (link)). Single-cell bone marrow RBC lysed cell suspension was obtained as described. Cells were resuspended in DMEM (Corning 10–013-CV) media supplemented with 50 µM β-mercaptoethanol (Sigma) and 20 µM OPP (Thermo Scientific C10456). Cells were incubated for 45 min at 37 °C and then washed with Ca²⁺ and Mg²⁺ free PBS. The samples were stained with biotin-labeled antibodies, followed by staining with fluorochrome-conjugated antibody cocktails, fixed and permeabilized using Cytofix/Cytoperm as described above. After permeabilization with Perm/Wash buffer, cells were resuspended in Click-iT Plus Reaction Cocktail (Thermo Scientific C10456) containing azide conjugated to Alexa Fluor 488 for 30 min at room temperature, washed once with Click-iT Reaction Rinse Buffer and resuspended in Perm/Wash buffer. Flow cytometry was performed with Cytek Aurora and data analysis was done with FlowJo Software (v9, v10).

Folgado-Marco V., Ames K., Chuen J., Gritsman K, & Baker N.E. (2023). Haploinsufficiency of the essential gene Rps12 causes defects in erythropoiesis and hematopoietic stem cell maintenance. eLife, 12, e69322.

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Ionizing Radiation-Induced Cell Cycle Arrest

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Cells were pulsed with 5-ethynyl-2′-deoxyuridine (EdU, Jena Bioscience) and subsequently exposed to ionizing radiation generated by Precision X-RAD 225XL (dose 3, 6, or 10 Gy) and were grown for further 20 h in the presence of nocodazole (250 ng/mL). Cells were stained with anti-pSer10-histone H3 (mitotic marker, Santa Cruz) and 4′,6-diamidino-2-phenylindole (DAPI as a marker of DNA content) and EdU was labeled with AlexaFluor 647 using Click-iT reaction (Thermo Scientific). Cells were analyzed by flow cytometry using LSRII (BD Biosciences) and FlowJo software (FlowJo). EdU-positive cells were assayed for progression through the G2 phase to mitosis (4n DNA content, pH3+). EdU-negative cells with 2n content were used for quantification of cells arrested in G1 checkpoint.

Burocziova M., Burdova K., Martinikova A.S., Kasparek P., Kleiblova P., Danielsen S.A., Borecka M., Jenikova G., Janečková L., Pavel J., Zemankova P., Schneiderova M., Schwarzova L., Ticha I., Sun X.F., Jiraskova K., Liska V., Vodickova L., Vodicka P., Sedlacek R., Kleibl Z., Lothe R.A., Korinek V, & Macurek L. (2019). Truncated PPM1D impairs stem cell response to genotoxic stress and promotes growth of APC-deficient tumors in the mouse colon. Cell Death & Disease, 10(11), 818.

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EdU Incorporation in Arrestin Knockout Cells

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HEK293 arrestin-2/3 knockout cells were either left untransfected or transfected with HA-arrestin-3 (1 μg), FLAG-MELK¹⁻³⁴⁰ (1 μg), or a fixed ratio of the two plasmids for 46 h prior to treatment with 10 mM EdU (5-ethynyl-2’-deoxyuridine) for 2 h. Cells were then detached from support using 0.025% trypsin, and the protease was quenched with DMEM medium (10% FBS, 1% Pen/Strep). Cells were immediately washed with 3 mL of 1% bovine serum albumin (BSA) in PBS and centrifuged at 700×g. The pellet was then fixed for 15 min at room temperature with 4% paraformaldehyde in PBS and the wash step was repeated. A 100 μL saponin-based permeabilization and wash reagent was used to prepare the cells for the Click-iT™ reaction (ThermoFisher Scientific).
A Click-iT™ reaction mixture was prepared using PBS, copper protectant, reaction buffer additive, and picolyl azide fluorescent dye following manufacturer’s instructions (ThermoFisher Scientific). Cells were incubated with 500 μL of the mixture for 30 min prior to a final wash with saponin-based permeabilization and wash reagent. Cells were resuspended in 500 μL buffer and analyzed using flow cytometry. Flow analysis was performed using a 3-laser LSRII (BD Biosciences). Analysis of flow cytometry data was performed using FlowJo 10.1.5 (FlowJo LLC, Ashland, OR).

Perry N.A., Fialkowski K.P., Kaoud T.S., Kaya A.I., Chen A.L., Taliaferro J.M., Gurevich V.V., Dalby K.N, & Iverson T.M. (2019). Arrestin-3 interaction with maternal embryonic leucine-zipper kinase. Cellular signalling, 63, 109366.

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Pulse-Labeling Zebrafish Larvae with EdU

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The larvae were incubated with 500 μM EdU for 1 h in E3 water with 2% DMSO to facilitate EdU solubilization. After pulse labeling, larvae were rinsed with E3 water, anaesthetized with 0.2% tricaine and fixed in 4% PFA. The CLICK-IT reaction for EdU labeling was performed according to the manufacturer's instruction (Thermo Fisher Scientific).

Wang W., Hu Y.F., Pang M., Chang N., Yu C., Li Q., Xiong J.W., Peng Y, & Zhang R. (2021). BMP and Notch Signaling Pathways differentially regulate Cardiomyocyte Proliferation during Ventricle Regeneration. International Journal of Biological Sciences, 17(9), 2157-2166.

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Quantifying Zebrafish Cell Proliferation

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The larvae at 123 hpf were incubated with 500 μM of EdU for 1 h in E3 water containing PTU and 2% DMSO to facilitate EdU solubilization. After treatment, larvae were rinsed with E3 water, anesthetized with 0.2% tricaine, and fixed overnight in 4% paraformaldehyde (PFA). The CLICK-IT reaction for EdU labeling was performed according to the manufacturer’s instruction (Thermo Fisher Scientific).

Peng Y., Wang W., Fang Y., Hu H., Chang N., Pang M., Hu Y.F., Li X., Long H., Xiong J.W, & Zhang R. (2021). Inhibition of TGF-β/Smad3 Signaling Disrupts Cardiomyocyte Cell Cycle Progression and Epithelial–Mesenchymal Transition-Like Response During Ventricle Regeneration. Frontiers in Cell and Developmental Biology, 9, 632372.

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