Cells were seeded into 12-well plates at a density of 2×105 cells/well and treated with 4 µM Bay-299 for 48 hours. The Edu incorporation assay was performed according to the manufacturer’s instructions. Briefly, Edu (Thermo Fisher Scientific, Click-iT™ Plus Edu Alexa Fluor™ 647 Flow Cytometry Assay Kit, #C10635) was added to the cells at a final concentration of 10 µM. After incubation for 2 hours, cells were harvested, washed twice with PBS, and stained using the Click-iT™ Plus Edu Alexa Fluor™ 647 Flow Cytometry Assay Kit. The samples were analyzed with the BD FACSCANTO II system (BD Biosciences) to determine the percentage of Edu incorporation.
Click it plus edu alexa fluor 647 flow cytometry assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit is a cell proliferation and viability detection tool. It utilizes the Click-iT EdU (5-ethynyl-2'-deoxyuridine) labeling method to detect DNA synthesis, and the Alexa Fluor 647 fluorescent dye for flow cytometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (6 mentions)
Facscanto 2, by BD (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Cytoflex lx, by Beckman Coulter (4 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (4 mentions)
Lsr 2 flow cytometer, by BD (3 mentions)
Facscanto, by BD (3 mentions)
Facsaria 2, by BD (3 mentions)
Facsaria 3, by BD (3 mentions)
Fxcycle violet, by Thermo Fisher Scientific (3 mentions)
Anti cd8α, by Thermo Fisher Scientific (2 mentions)
Anti cd8β, by Thermo Fisher Scientific (2 mentions)
Anti vγ1 clone 2, by BioLegend (2 mentions)
Anti cd3, by Thermo Fisher Scientific (2 mentions)
7 aad, by Thermo Fisher Scientific (2 mentions)
Facs lsrii flow cytometer, by BD (2 mentions)
Fxcycle pi rnase staining solution, by Thermo Fisher Scientific (2 mentions)
Click it edu alexa fluor 488 flow cytometry assay kit, by Thermo Fisher Scientific (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Facsdiva software, by BD (2 mentions)
107 protocols using click it plus edu alexa fluor 647 flow cytometry assay kit
1
Edu Incorporation Assay for Cell Proliferation
2
EdU Incorporation Assay for Cell Cycle Analysis
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Cells were seeded in 6-well plates at a density of 1 × 105 cell/mL and incubated with EdU (10 µM) for 1 h (Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit, Invitrogen). After washing with PBS three times, cells were cultured in fresh complete media for 2, 4 and 8 h. They were then washed with 1% BSA, fixed in 70% ethanol and stored at −20 °C for at least 2 h. Next, cells were washed again with 1% BSA and incubated with 1 × Click-iT™ saponin-based permeabilization and wash reagent for 15 min at room temperature. They were subsequently incubated with Click-iTEdU reaction buffer at room temperature for 30 min protected from light. For propidium iodide staining, cells were then resuspended in propidium iodide/Triton X-100 (9036-19-5, Merck) staining solution with RNase A (740505, Cultek) (0.1% (v/v) Triton X-100, 2 mg RNase, 500 µM PI) for 30 min at room temperature. Cells were then subjected to flow cytometry (Coulter EPICS (R) XL Flow Cytometry System). Forward and side scatter area gating were used to identify singlets. EdU incorporation was detected using 633/635 nm excitation with a red emission filter (660/20 nm). The percentages of cells in each cell cycle phase were determined using FlowJO software.
3
Measuring Intestinal Cell Proliferation
4
LSK BM Cell Proliferation Assay
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LSK BM cells were incubated with 10 μM EdU for 5 and 8 h, followed by fixation, permeabilization, and incubation with fluorescently conjugated antibody and 4’,6-diamidino-2-phenylindole according to the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay kit protocol (Invitrogen, Cat No. C10634).
5
Tissue-Resident Mononuclear Cells Turnover
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CTRL (Bimfl/fl) and LysMCreBimfl/fl mice were given 2 mg EdU (Santa Cruz Biotechnology, Inc.) through subcutaneous injections daily for 49 d. To examine turnover rate and proliferation in tissue-resident monocytes/macrophages, mice were sacrificed at several time points (days 35, 49, and 63). Single-cell suspensions from bone marrow, blood, spleen, and kidney were stained with fluorochrome-conjugated antibodies. EdU staining was assayed with intracellular flow cytometry using a Click-iT Plus EdU Alexa Fluor647 Flow Cytometry Assay kit (Invitrogen) according to the manufacturer’s protocol. Each tissue came along with an FMO control to identify the EdU-positive cells. Stained cells were acquired on an LSRII flow cytometer. Compensation and data were analyzed using FlowJo software (Tree Star).
6
Apoptosis and Cell Cycle Analysis of ESCs
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ESCs were digested and collected for the apoptosis assay, which was performed according to the manufacturer’s instructions of Annexin V-FITC Apoptosis Detection Kit (Beyotime). For the cell cycle analysis, 10 μmol/L EdU was added to the culture medium 2 h before the cells were harvested. The EdU labeling was then performed following the instructions of Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen). The percentages of apoptotic cells and S-phage cells were determined by fluorescence-activated cell sorting (FACS) and the results were analyzed through CytExpert software.
7
Cell Proliferation Assay via EdU Labeling
8
Cell Cycle Analysis by EdU and PI Staining
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For dual EdU and propidium iodide (PI) staining for cell cycle analysis, cells were trypsinised, pelleted and resuspended in PBS at a density of 1.5 × 106 cells/ml. Ethanol was slowly added to the cell suspension to a concentration of 70% to fix and permeabilise the cells, which were incubated on ice for a minimum of 30 min or stored at 4°C for up to 2 weeks. EdU staining was performed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen, CatNo C10634) following the manufacturer’s instructions. Cells were stained in a solution containing 1 μg/ml PI and 4 μg/ml RNase A in PBS at 2 × 106 cells/ml for a minimum of 30 min at RT. Cell cycle analysis was performed on a LSR Fortessa analyzer (BD Biosciences) and analyzed using FlowJo software.
9
Quantifying Cell Proliferation in HFOs
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Cell proliferation was measured using a Click-iT® plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit (Invitrogen, Thermo Fisher Scientific, cat#C10634). Briefly, 72 h after cytokines stimulation HFOs were incubated with 5-ethynyl-2′-deoxyuridine (EdU) for 4 h. Subsequently cells were harvested in cell recovery solution (Corning®, cat#354253) for 30 mins on ice and single cells were generated by incubating 3D cultures in TrypLE Express (Invitrogen, cat#12605036) for 10 mins at 37°C. Cells were fixated in 4% formaldehyde and permeabilized with saponin, stained for 30 mins at room temperature (RT) protected from light, and acquired by flow cytometry (FACS Diva, BD Bioscience). Results were analyzed using FlowJo software version 10.3 (Treestar, Ashland, OR, USA).
10
Cell Cycle Analysis by Dual Nucleotide Pulse
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Cell suspensions were resuspended in PBE and incubated on ice for 30 min with fluorescently labeled antibodies (Table S2 ) along with 1 mg/ml anti-CD16/32 (24G2; eBioscience).
For detection of cells in early S of the cell cycle, we performed dual nucleotide pulse and staining as previously described (Gitlin et al., 2014 (link)). Briefly, mice were injected i.v. with 1 mg of EdU (A10044; Thermo Fisher Scientific) and 1 h later with 2 mg BrdU (B5002; Sigma). 30 min after the second injection, LNs were harvested, and single-cell suspensions were prepared. After cell surface receptor staining as described above, cells were fixed and permeabilized using BD Cytofix/Cytoperm fixation and permeabilization solution and BD Cytoperm Permeabilization Buffer PLUS, respectively. EdU and BrdU incorporation into DNA was assayed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) and FITC BrdU Flow Kit (BD), respectively.
For single-cell sorting, cells were stained as above and index-sorted directly into 96-well plates containing Buffer TCL (Qiagen) supplemented with 1% β-mercaptoethanol using a BD FACS Aria II. Each plate contained all conditions assayed in each replicate. Cells were washed, filtered, and resuspended in PBE before analysis or sorting on BD FACS LSR II, FACS Symphony, or FACS ARIA II cytometers. All data were analyzed using FlowJo software v.10.
For detection of cells in early S of the cell cycle, we performed dual nucleotide pulse and staining as previously described (Gitlin et al., 2014 (link)). Briefly, mice were injected i.v. with 1 mg of EdU (A10044; Thermo Fisher Scientific) and 1 h later with 2 mg BrdU (B5002; Sigma). 30 min after the second injection, LNs were harvested, and single-cell suspensions were prepared. After cell surface receptor staining as described above, cells were fixed and permeabilized using BD Cytofix/Cytoperm fixation and permeabilization solution and BD Cytoperm Permeabilization Buffer PLUS, respectively. EdU and BrdU incorporation into DNA was assayed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) and FITC BrdU Flow Kit (BD), respectively.
For single-cell sorting, cells were stained as above and index-sorted directly into 96-well plates containing Buffer TCL (Qiagen) supplemented with 1% β-mercaptoethanol using a BD FACS Aria II. Each plate contained all conditions assayed in each replicate. Cells were washed, filtered, and resuspended in PBE before analysis or sorting on BD FACS LSR II, FACS Symphony, or FACS ARIA II cytometers. All data were analyzed using FlowJo software v.10.
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