Cells were washed once in ice-cold phosphate-buffered saline (PBS), fixed in 70% ethanol at −20°C for 2 h, resuspended in PBS solution containing 4 μg/ml of propidium iodide and 0.1 mg/ml of RNase A, and incubated overnight before quantification of propidium iodide fluorescence. For Edu incorporation assays, cells were treated with 2 μM of Click-iT EdU (5-ethynyl-2’-deoxyuridine) reagent (Click-iT™ Plus EdU Alexa Fluor™ 647, Invitrogen) for 1 h before harvesting and fixation in 70% ethanol at −20°C overnight. Cells were then incubated for 30 min with the Click-iT EdU reaction mixture, as indicated by the manufacturer, followed by another 30 min incubation with the propidium iodide and RNase A containing PBS solution. Fluorescence detection was performed using CytoFLEX flow cytometer (Beckman Coulter) and cell distribution in the cell cycle was determined with the FlowJo™ Software (Becton Dickinson) after gating out cell debris signals.
Click it plus edu alexa fluor 647
Manufactured by Thermo Fisher Scientific
The Click-iT™ Plus EdU Alexa Fluor™ 647 is a fluorescent labeling reagent used for the detection of newly synthesized DNA in cells. It is designed to enable the visualization and quantification of cell proliferation through the incorporation of the thymidine analog EdU (5-ethynyl-2'-deoxyuridine) into the DNA of dividing cells.
Automatically generated - may contain errors
Lab products found in correlation
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Facscalibur, by BD (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20 cytometer, by BD (1 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
Accumax, by Merck Group (1 mentions)
Fxcycle violet stain, by Thermo Fisher Scientific (1 mentions)
Attune nxt flow cytometer, by BD (1 mentions)
4 protocols using click it plus edu alexa fluor 647
1
Cell Cycle Analysis and Proliferation Assay
2
Synchronization and Cell Cycle Analysis of HUVECs
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HUVECs were synchronized by a 48 h starvation in EBM2 containing 0.5% FBS. Next, cells were stimulated or not with BMP10 (10 ng/mL) overnight in EBM2 supplemented with 0.5% or 5% FBS. For the analysis of cell cycle progression, actively proliferating cells were detected using Click-iT™ Plus EdU Alexa Fluor™ 647 (Invitrogen) according to the manufacturer’s instructions with minor modifications. Thirty thousand cells were analyzed on the FACS Calibur (BD Biosciences), and data were analyzed using FCS Express.
3
Multiparametric Flow Cytometry of 3D Tumor Spheroids
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The proportions, survival, and cell cycle of each population from spheroids were measured by flow cytometry. Mixed unirradiated or irradiated spheroids (seeded 1:1 parental:RR, 6–8 pooled/group) were incubated with EdU (10 µM final concentration) 12 h prior to dissociation, dissociated (100 μL Accumax, Millipore) for 20 min at 37 °C, washed with phosphate-buffered saline (PBS), centrifuged (300 × g, 5 min), and incubated with efluor-780 (1 μL/mL PBS; ThermoFisher Scientific) for 30 min on ice in the dark to distinguish live/dead cells. After washing in PBS, samples were fixed for 10 min in IC Fixation Buffer (ThermoFisher Scientific), and permeabilized and stained with Click-iT Plus EdU Alexa Fluor 647 (ThermoFisher Scientific) according to manufacturer’s instructions. Following a wash in 1× saponin, cells were incubated 30 min with FxCycle Violet Stain (1:1000, 300 µL of 1× saponin; ThermoFisher Scientific) before being run on the BD LSR Fortessa X-20 Cytometer or the Attune NxT Flow Cytometer using the 405, 488, 561, and 633 lasers. Data were analysed using FlowJo (Treestar, Inc.) as described in Supplementary Figs. 2 and 4 .
4
Quantifying Proliferative Endothelial Cells
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To quantify proliferative endothelial cells, we first performed IHC for pan-endothelium (rabbit anti-ERG, 1:100, ab214341, Abcam) and EMCN, which stains for capillary and venous endothelium. Following IHC, we performed a click chemistry-based EdU proliferation assay (Click-iT Plus EdU Alexa Fluor 647, C10640, Thermo Fisher Scientific). Pregnant dams were injected intraperitoneally at the desired time point with 50 gm/kg of EdU, and embryos were harvested 4 h following injection. We captured five random areas on each tissue section from each embryo. We counted the percentage of EdU+ cells in arteries, veins and capillaries. Briefly, we counted all ERG+ cells to determine the total endothelial cells in the field of view. We classified EMCN−/ERG+ cells as arterial endothelial cells and EMCN+/ERG+ cells as either capillary or venous endothelial cells. We differentiated venous cells from capillary cells by tracing a subset of cells which formed a lumen. Among these, EdU+ cells were counted to determine the percentage of proliferative cells in each subtype.
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