Click it plus edu alexa fluor 647 flow cytometry kit

Mice were injected intraperitoneally with 1 mg EdU (Lumiprobe) 2 h before tissue collection for assessment of cell proliferation. EdU detection was carried out using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Kit (Thermo Fisher Scientific) after surface staining and fixation and permeabilization. Intranuclear staining of Ki-67 was performed in parallel to EdU detection. Cells were analyzed using flow cytometry as described above.

Cells were plated in 6-well plates (TPP), and incubated for 24 h, before treatment with palbociclib, ribociclib or the corresponding vehicle. After 94 h of treatment, 10 μM EdU was added to the cell culture media for 2 h. The cells were then collected and washed twice with PBS. Click-it reactions were performed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Kit (ThermoFischer Scientific) according to the manufacturer's recommendations. The cells were then counterstained with propidium iodide for 30 min. The cell cycle profile was generated using a CyAn ADP 9C analyser (Beckman Coulter). The analysis was performed with the Flowjo software (LLC). Cell cycle was studied on technical duplicates, in three independent experiments.

Cell cycle analysis was performed with FX Cycle violet stain (Invitrogen, Carlsbad, CA, F10347), and S phase analysis was conducted with EdU labeling (Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Kit®, Thermo Fisher Scientific, Waltham, MA, USA). Samples were treated on a Gallios flow cytometry system (Beckman Coulter, Brea, CA, USA)³³ . Data were analyzed with FCS Express 6 + software (DeNovo Software®, Glendale, CA, USA).

10μM EdU were added to cells 48h post-DOX induction. After 1h30, they were harvested and washed twice with PBS. Cells were incubated for 30 min with chicken anti-GFP (Abcam, ab13970; 1/2000) and then 30 min with Alexa Fluorophores A488 coupled secondary antibodies (Thermo Fisher Scientific; 1/2000). After three PBS washes, click-it reactions were performed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Kit (Thermo Fischer Scientific) according to the manufacturer’s recommendations. Cells were counterstained with DAPI for 30 min. Sample measurements were done on a BD FACSAria Fusion flow cytometer (BD Biosciences) and with FACSDiva Software (v8.0.1, BD Biosciences). Alexa Fluor 488 signal was measured by 488nm laser excitation with a 530/30 bandpass filter, Alexa Fluor 647 by 633nm laser with a 670/30 nm bandpass filter and DAPI by 405nm laser with a 450/40 nm bandpass filter. Data analysis was performed with FlowJo v10.7.1 Software (BD Biosciences). Cells were identified on a Side Scatter (SSC) vs Forward Scatter (FSC) dot plot and cell debris and aggregates were excluded from analysis based on FSC signals. Cell cycle was studied on three independent cell lines, in three independent experiments.

Cell proliferation assays were performed by directly measuring DNA synthesis using an Alexa Fluor 647 click-iT plus EdU flow cytometry kit (Thermo Fisher) according to the manufacturer’s instructions. 5-Ethynyl-2′-deoxyuridine (EdU, 10 μM) was added to the culture medium for a 12-h incubation in a humidified atmosphere at 37 °C with 5% CO₂ at the end of the 12-h coincubation (0, 10, 20, 40, 80 µg/mL HcESPs with T cells) period. Subsequently, T cells were fixed with 4% paraformaldehyde in PBS and permeabilized with Click-iT saponin-based permeabilization and wash reagent, followed by Click-iT reaction to couple EdU with the Alexa Fluor 647 dye. After two washes with 3 mL of 1% BSA in PBS, T cells were treated with 7-AAD Staining Solution (BD Biosciences), and standard flow cytometry methods were used to determine the percentage of S-phase cells in the population. Each experiment consisting of three replicates was performed in triplicate.

Cell proliferation analysis was determined using the Alexa Fluor 647 Click-iT plus EdU flow cytometry kit (Thermo Fisher Scientific) via the measurement of DNA synthesis directly based on the manufacturer’s instructions. After 12 h co-incubation, the cell culture was incorporated with 5-ethynyl-2′-deoxyuridine (EdU, 10 μM) for another 12 h incubation. Subsequently, T cells were harvested, fixed with 4% paraformaldehyde in PBS and permeabilized using the Click-iT saponin-based reagent, followed by Click-iT reaction to coupled EdU with Alexa Fluor 647 dye. After three washes with 3 ml of 1% BSA in PBS, T cells were treated with 7-AAD staining solution (BD Biosciences). Flow cytometry was used for the determination of EdU⁺ cells in the population. Each experiment consisting of three replicates was run in triplicate.

The detection of cell proliferation was performed by measuring DNA synthesis directly using the Alexa Fluor 647 click-iT plus EdU flow cytometry kit (Thermo Fisher) according to the manufacturer's instruction. At the end of the 12 h co-incubation period, 5-ethynyl-2'-deoxyuridine (EdU, 10 μM) was added to the culture medium for another 12 h incubation in a humidified atmosphere at 37°C with 5% CO2. Subsequently, T cells were harvested, fixed with 4% paraformaldehyde in PBS and permeabilized using Click-iT saponin-based reagent, followed by Click-iT reaction to couple EdU with Alexa Fluor 647 dye. After being washed with 3 mL of 1% BSA in PBS, T cells were treated with 7-AAD staining solution (BD Biosciences). Flow cytometry was used for determination of the percentage of EdU⁺ cells in the population. Three independent experiments were performed and each experiment was run in triplicate.