Click it edu alexa fluor 488 flow cytometry assay kit

Cells were trypsinized and collected by centrifugation. The cells were washed twice with PBS and fixed with cold 70% ethyl alcohol at -20 °C for 1 hour. After washed with PBS, the cells were treated RNase A, stained 20 µg/mL propidium iodide at 37°C for 30 min and analyzed with CytoFLEX (CytoFLEX; Beckman, Brea, CA,US) flow cytometer.
Click-iT^® EdU Alexa Fluor^® 488 Flow cytometry assay kit (C10632; Invitrogen Waltham, MA, US) was used to cell cycle analysis according to the manufacturer's protocol. EdU staining and flow cytometry cells were incubated with 10 µM EdU for 2 h. Harvested cells were fixed with 4% paraformaldehyde at RT for 15 min and permeabilized by saponin-based permeabilization buffer for 15 min. The cells were digested with a reaction solution containing Alexa Fluor^® 488 at was added at room temperature, in the dark, for 30 min and determined using CytoFLEX (CytoFLEX; Beckman, Brea, CA, US) flow cytometer.

Cell cycle analyses were performed using Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (Invitrogen) according to manufacturer instructions.

For testing cell cycle, 1x10⁶ T cells in 1 mL DMEM complete media were incubated with 5 µM Vybrant™ DyeCycle™ Violet Stain (Thermo Fisher) at 37˚C for 30 min. And then 5 µl 7AAD were added and incubated for 10 min prior to analysis. Total 25,000 cells were analyzed per measurement. The same forward and side scatter gates were applied to each sample, and within that gate we measured the intensity of vibrant cell cycle dye. Samples were analyzed on a flow cytometer using 405 nm excitation and 440 nm emission. To compare the growth rates of B16 WT and apoE^-/- cells, the Click-iT Edu Alexa Fluor 488 flow cytometry assay kit was used in conjunction with the FxCycle Violet stain from Invitrogen (Carlsbad, CA). 50,000 cells were plated per well of a 6 well-plate and cell cycle was analyzed at 48 hr as per the manufacturer’s directions.

800 000 DU145 cells were plated in 10-cm diameter dishes for 24 h before treatment with polyamides for an additional 24 h. Cells were pulsed with 10-μM ethynyldeoxyuridine (EdU) 30 min before harvest to estimate the rate of DNA synthesis. Cells were trypsinized and pelleted at 300 × g with cell culture supernatant. Following overnight fixation in 70% ethanol, the cells were rehydrated in 1% BSA/PBS and processed with the Click-it EdU Alexa Fluor 488 Flow Cytometry assay kit (Invitrogen) using half the recommended A488 reagent. After overnight treatment with 0.2-mg/ml RNase A in 1% BSA/PBS, the cells were stained for DNA content with 7-aminoactinomycin D and analyzed on a FACSCalibur (Becton-Dickinson) instrument. The data were analyzed using FlowJo v9.5.3 (TreeStar) and are representative of two trials. Monoparametric, propidium iodide, flow cytometry was also used to evaluate the effect of polyamides 1 and 2 on cell cycle distribution. DU145 cells were treated with 1–100 μM of polyamide 1 or 0.1–10 μM of polyamide 2 for 48 h. The effect of PI3 kinases on cell cycle distribution was measured by treating DU145 cells with 10-μM polyamide 1 or 1-μM polyamide 2 as well as 2-mM caffeine, 4- or 10-μM NU6027 (Calbiochem) and 4- or 10-μM KU55933 (Calbiochem) for 36 h. Data were analyzed using FlowJo and fitted to the Watson (Pragmatic) model. The data are representative of two trials.