Click-iT™ EdU Alexa Fluor™ 555 Imaging Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 8 × 104 cells per well were platted for the assay	- Cells were labelled for 2 h 30 with 10 μm EdU.

Upstream tips
- 8 × 104 cells per well were platted for the assay
Protocol tips
- Cells were labelled for 2 h 30 with 10 μm EdU.

Publication protocol

The Click-iT EdU cell proliferation assay (Invitrogen, Ref. C-10338) was used to detect and quantify newly synthetized DNA into cells growing on different PE coatings, including the mono or multilayers PE coatings, in 6-well plates. Hydroxyurea 10 mm (Sigma, Ref. H8627) was used as a positive control for cell cycle inhibition since it blocks the cell cycle in G1/S phase and thus inhibits cell proliferation. Fibronectin (2 μg/ml in PBS+) and no-coating conditions were used as positive controls of proliferation. The influence of the PE films on DNA synthesis was evaluated 72 h after having plated the cells at 8 × 104 cells per well. Cells were then pulse labelled for 2 h 30 with 10 μm EdU. The modified nucleoside EdU (5-ethynyl-2′-deoxyuridine) provided in the kit is incorporated into DNA during active DNA synthesis. Immediately after exposure to EdU, cells were trypsinized (Trypsin-EDTA (8 min, 37 °C)) and fixed (PFA 4%, 15 min at RT, in PBS+) and then stained with the Click-it EdU detection reagent (30 min, RT, protected from light) in order to detect EdU with the Alexa Fluor azide 555 dye according to the manufacturer's instructions. Centrifugation (5 min/600 g/RT) was then performed, followed by a wash with PBS and 3% BSA (Bovine Serum Albumin). The supernatant was discarded and the pellet was re-suspended in PFA 2% in PBS+ and kept at 4 °C before FACS analysis. FACS (Becton Dickinson LSR II) flow cytometer was used to scan and collect the cell data. The FACS Diva software was used to generate flow cytometry data and to estimate the proportion of proliferative cells at 72 h. Statistically significant differences between three independent experiments were identified using the Mann–Whitney test.

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Manufacturer protocol

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