5 ethynyl 2 deoxyuridine (edu)

Cells were labeled briefly with 20 µM Edu (Click Chemistry Tools) for 2 h, incubated at 37 °C, fixed with 3.7% formaldehyde for 15 min, permeabilized in 0.5% Triton-X 100 in PBS. Each step was followed by a wash using 3% BSA in PBS, centrifuged on a table-top centrifuge at 2000g for 1 min. The cells labeled with Edu were incubated in the Click-IT reaction mix (100 mM Tris-Cl (pH7.5), 3 mM CuSO₄, 50 mM Ascorbic Acid, 2.5 µM Alexa-647 Azide) for 30 min in dark. The cells in specific cell cycle stages were detected using 3 µM (892 ng/ml) DAPI in PBS. All samples were analyzed in triplicates using BD LSR-Fortessa flow cytometer and BD FACS-Diva 8.0.1 software.

If measuring 5-ethynyl-2'-deoxyuridine (EdU) incorporation, cells were pulsed with 50 μM EdU (Cayman Chemical Cat# 20518) in growth media for 8 min prior to fixation and pre-extraction, unless otherwise stated. EdU is incorporated throughout the EdU pulse, such that incorporated EdU reflects the average rate of DNA synthesis over the length of the pulse. Thus an 8 min short EdU pulse is more reflective of the instantaneous DNA synthesis rate compared to a longer pulse such as 1 h. After blocking cells (prior to primary antibodies), cells were washed once with PBS, and then a click reaction^{67 (link)} was performed in 2 mM CuSO₄, 20 mg/mL sodium ascorbate in TBS (Tris 50 mM, NaCl 150 mM pH 8.3) with 3 μM AFDye 488 picolyl azide (Click Chemistry Tools Cat# 1276) or AFDye 647 picolyl azide (Click Chemistry Tools Cat# 1300) for 30 min, followed by a PBS wash.