Click it edu flow cytometry cell proliferation assay

Neural precursor cells were cultured under proliferation conditions in PDL/laminin-coated 6-well plates with recombinant mouse PF4 protein diluted in sterile 0.9% sodium chloride. Control cells were cultured with an equal volume of sterile 0.9% sodium chloride. After 48 h, the cells were incubated with EdU (final concentration 10 μM) for 2 h and the Click-iT® EdU Flow Cytometry Cell Proliferation Assay (Thermo Fisher Scientific cat# C10634) was performed according to the manufacturer’s instructions. The cells were also incubated with Hoechst 33342 (1:5000 in saponin-based permeabilization and wash reagent) for 10 min in the dark. They were then transferred into a flow cytometry tube and analysed using an LSRII flow cytometer and FACSDiva software (v9.0; BD Biosciences) on the same day. For this, viable cells were first determined using forward scatter and side scatter. Next, doublets were excluded from single cells by plotting Hoechst-width against Hoechst-area. Finally, to define the cell cycle phases, the DNA content (Hoechst-area) was plotted against the EdU signal. A total of 50,000 single cell events were recorded by flow cytometry. Data analysis was performed using FlowJo software (BD, v10.8.1). A total of seven independent experiments were performed with three technical replicates each.

For EdU/DAPI FACS analysis, Pole4^+/+ and Pole4^−/− MEFs (passage 3) were labeled and processed using the Click-iT EdU Flow Cytometry Cell Proliferation Assay (Thermo Fisher). Cells were pulse labeled for 30 min with 10 μM EdU and fixed in 4% paraformaldehyde, before being permeabilized in PBS-Triton 0.5% and washed in 1% BSA. Cells were then resuspended in Click-iT reaction cocktail containing Alexa Fluor 488 Azide and incubated for 30 min at R.T. After being washed, cells were finally counterstained for DNA content by DAPI (1 mg/ml) and analyzed using a Flow cytometry analyzer LSRII (Becton Dickinson).
For BrdU/DNA content analysis of human dermal fibroblasts derived from control or POLE1 mutant patients, cells were pulse labeled with 10 μM BrdU (SIGMA) for 30 min, washed and released or not in normal media for 4, 8, 12 or 16 hours to analyze S-phase progression. Cell were trypsinized, pelleted and fixed in ice-cold 70% ethanol. After washing, cells were resuspended in 500 mL of 2M HCl and incubated at R.T for 30 min with occasional mixing. Cells were pelleted, washed to remove excessive acid and incubated with anti-BrdU antibody for 20 min at R.T. and rabbit anti-mouse FITC-conjugated (DAKO) for 20 at R.T. in the dark. Cells were finally washed, resuspended in PBS-T containing RNase A and Propidium Iodide and analyzed using a Flow cytometry analyzer LSRII (Becton Dickinson).

NPCs were cultured in proliferation medium with recombinant mouse XCL1 protein or 0.1% BSA. After 48 h, the Click-iT^® EdU Flow Cytometry Cell Proliferation Assay (Thermo Fisher Scientific) was performed according to the manufacturer’s instructions. Subsequently, analysis was performed using an Aria III flow cytometer (BD Biosciences) and the FlowJo software. In total, the experiment was performed four times with two technical replicates each.

For EdU/PI Flow Cytometry, cells were labeled for 30 min with 10 μM EdU, fixed in 4% PFA, permeabilized in PBS-Triton 0.3% and washed in 1% BSA before samples were processed using the Click-iT EdU Flow Cytometry Cell Proliferation Assay (Thermo Fisher) with Alexa Fluor 488. DNA was counterstained with Propidium Iodide (10 μg/mL). Newly synthesized DNA (EdU) and DNA content (PI) were detected using an LSRII (Becton Dickinson). Gating of single cells and cell cycle analysis was performed manually using FlowJo (TreeStar).

A proliferation assay was performed using Click-It EdU Flow Cytometry Cell Proliferation Assay (ThermoFisher Scientific). For 2D proliferation assays, 2.5 × 10³ ECs were plated in six well plates and starved in serum-free M199 supplemented with 0.1% BSA for 10 hr. Cell proliferation was induced by the addition of 20 ng/ml VEGF-A to the culture medium, and maintained for additional 18 hr. 10 µM of 5-ethynyl-2′-deoxyuridine (EdU) was added 2 hr prior to cell harvesting. For cell proliferation measurement in spheroids, 10 µM EdU was added 2 hr prior to spheroids collection. EdU detection was performed according to the manufacturer's protocol and samples were analyzed in a CyAn ADP flow cytometer with Summit five software (Beckman Coulter, Brea, CA, USA).

Neural precursor cells were cultured under proliferation conditions in PDL/laminin-coated 6-well plates with recombinant mouse PF4 protein diluted in sterile 0.9% sodium chloride. Control cells were cultured with an equal volume of sterile 0.9% sodium chloride. After 48 h, the cells were incubated with EdU (final concentration 10 μM) for 2 h and the Click-iT® EdU Flow Cytometry Cell Proliferation Assay (Thermo Fisher Scientific cat# C10634) was performed according to the manufacturer’s instructions. The cells were also incubated with Hoechst 33342 (1:5000 in saponin-based permeabilization and wash reagent) for 10 min in the dark. They were then transferred into a flow cytometry tube and analysed using an LSRII flow cytometer and FACSDiva software (v9.0; BD Biosciences) on the same day. For this, viable cells were first determined using forward scatter and side scatter. Next, doublets were excluded from single cells by plotting Hoechst-width against Hoechst-area. Finally, to define the cell cycle phases, the DNA content (Hoechst-area) was plotted against the EdU signal. A total of 50,000 single cell events were recorded by flow cytometry. Data analysis was performed using FlowJo software (BD, v10.8.1). A total of seven independent experiments were performed with three technical replicates each.