Edu flow cytometry assay kits

The cell proliferation rates were detected by EdU Flow Cytometry Assay Kits purchased from Life Technologies (Carlsbad, CA, USA). SKM-1 cells or primary CD34 + cells were treated with 10 μM EdU for 1 h and assessed according to the recommended staining protocol. Cells labelled with Alexa Fluor^® 647 azide were analysed on a flow cytometer using 633 nm excitation and a 660/20 nm bandpass emission filter.
Cells were counted using a haemocytometer. MDS cells from different time points were collected and resuspended in 1 ml of PBS. One part of 0.4% trypan blue and one part of cell suspension were mixed. A drop of the trypan blue/cell mixture was applied to a haemocytometer. The unstained (viable) cells were counted under a microscope in four 1 × 1-mm squares of one chamber, and the average number of cells per square was determined. The cell count was determined as follows: average cell count per square × dilution factor × 10 ^ 4 = cell count per ml.

DNA synthesis was measured using 5-ethynyl-2´-deoxyuridine (EdU) Flow Cytometry Assay Kits (Life Technologies, Tokyo, Japan). Experiments were performed according to the manufacturer’s instructions. Briefly, 3 × 10⁶ cells were seeded into 10 cm dish and incubated for 24 hours at 37°C with or without 1 μg/ml tetracycline (Invitrogen). Cells were incubated for 2 hours with 20 μM EdU. Subsequently, cells were processed using the Click-iT EdU Flow Cytometry kit. EdU incorporation was measured by Flow cytometer (Gallios Flow Cytometer, Beckman Coulter).