Gata4loxP(Gata4tm1.1Sad), Gata4−(Gata4tmo1Eno), ShhCre(Shhtm1(EGFP/cre)Cjt/J), Gata4flbio/flbio(Gata4tm3.1Wtp), Rosa26BirA(Gt(ROSA)26SorTm1(birA)Mejr), and CD-1 (Charles River Laboratories, Wilmington, MA) mice were used.15 (link)–19 (link) Embryos were obtained by timed mating with a vaginal plug at noon considered E0.5. Genotypes were determined by PCR with tail tip, ear punch, or yolk sac gDNA (Primers, Table 1 ). For proliferation studies, 200 μg of 5-ethynyl-2′deoxyuridine (EdU, Invitrogen, Carlsbad, CA) was administered to pregnant mice by intraperitoneal injection 30 min before euthanizing. EdU incorporation was detected with the Click-it EdU Alexa-Fluor kit (Invitrogen, Carlsbad, CA). The Medical College of Wisconsin’s Animal Care Committee approved all animal procedures.
Click it edu alexa fluor kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Click-iT EdU Alexa Fluor kit is a fluorescent labeling system designed for the detection and quantification of newly synthesized DNA in proliferating cells. It utilizes a chemical reaction between an alkyne-modified nucleoside (EdU) and a fluorescent azide dye (Alexa Fluor) to label DNA during the S-phase of the cell cycle.
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8 protocols using click it edu alexa fluor kit
1
Cardiac Development Gene Analysis
2
Genetic Manipulation of Cardiac Development
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Gata4loxP(Gata4tm1.1Sad), Gata4-(Gata4tmo1Eno), ShhCre(Shhtm1(EGFP/cre)Cjt/J), Gata4flbio/flbio(Gata4tm3.1Wtp), Rosa26BirA(Gt(ROSA)26SorTm1(birA)Mejr), and CD-1 (Charles River Laboratories, Wilmington, MA) mice were used.15 , 16 , 17 , 18 , 19 Embryos were obtained by timed mating with a vaginal plug at noon considered E0.5. Genotypes were determined by polymerase chain reaction (PCR) with tail tip, ear punch, or yolk sac genomic DNA (Table 1 , primers). For proliferation studies, 200 μg of 5-ethynyl-2’deoxyuridine (EdU) (Invitrogen, Carlsbad, CA) was administered to pregnant mice by intraperitoneal injection 30 minutes before euthanizing. EdU incorporation was detected with the Click-it EdU Alexa-Fluor kit (Invitrogen). The Medical College of Wisconsin’s Animal Care Committee approved all animal procedures.
3
Immunofluorescence Staining of EdU and CYP11A1
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For EdU or CYP11A1 immunofluorescence staining, SLCs were plated in a 24-well plate containing a TC-treated round cover slide (WHB Scientific, Shanghai, China; Cat#, WHB-24-CS). Cells were expanded and differentiated by a routine procedure. SLC proliferation was determined by the Click-it® EdU Alexa Fluor Kit (Invitrogen, USA) according to the manufacturer’s instructions. In brief, the EdU-labeled cells were fixed with 4% paraformaldehyde for 30 min at room temperature followed by breaking the membrane with 0.5% Trion X-100. After being washed with PBS, the cells were treated with Click-it reagents to reveal nuclear EdU incorporation, followed by 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining of the nucleus. For CYP11A1 staining, the cells exposed to midazolam (5 and 30 μM) for 21 days were first fixed with 4% paraformaldehyde for 30 min, followed by incubation with primary antibodies (1:100) at 4°C overnight in darkness. After being washed with PBS, the cells were stained with fluorescent secondary antibodies for 1 h, followed by DAPI staining of the nucleus. The EdU+ cells and total nuclei stained by DAPI were counted under a ×20 objective.
4
Labeling Proliferating Cells in Mice
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Adult mice 1–3 months old were injected with 12.5 μg/g of body weight EdU (0.5 mg/ml DMSO stock) dissolved in 200 μL water. Mice were killed 2 h after injection and processed for FACS or for cryosections as described above. EdU detection with Click-iT EdU Alexa Fluor Kit was performed as described in the manual (Invitrogen).
5
Visualizing DNA Synthesis with EdU
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Ethynyl deoxyuridine (EdU) was dissolved in DMSO and added to the medium at a final concentration of 20 μM. Cells were fixed after 1 h, and EdU detection was performed by the Click-IT EdU Alexa Fluor kit from Thermo Fisher (C10337).
6
EdU Incorporation Analysis in Mice
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EdU incorporation was performed using a Click-iT EdU Alexa Fluor kit (C10419, ThermoFisher) according to the manufacturer's instruction. Briefly, 5 WT and 6 Eaf2−/− mice (10 weeks old) were immunized with NP–CGG and 12 days and 13 days later injected intraperitoneal (i.p.) with 200 μl of (1 mg ml−1) EdU. EdU incorporation was analysed on day 14 by FACS.
7
EdU Incorporation Analysis in Mice
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EdU incorporation was performed using a Click-iT EdU Alexa Fluor kit (C10424, ThermoFisher) according to the manufacturer’s instruction. Briefly, WT and FcμR−/− mice (12 weeks old) were injected intraperitoneal (i.p.) with 200 µl of (1 mg/ml) EdU. EdU incorporation was analyzed 24 h later by FACS.
8
Cell Cycle Analysis using EdU Labeling
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The cell cycle was analyzed using the Click-iT™ EdU Alexa Fluor™ kit (Thermo Fisher Scientific) according to the manufacturer’s protocol 48 h after transfection. The cells were cultured for 2 h with a 10 μM of EdU (5-ethynyl-2′-deoxyuridine). The cell cycle was measured by flow cytometry–BD FACSCelesta (BD Bioscience), and data were analyzed by Flowlogic software (Inivai Technologies).
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