Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Cells are labelled with EdU for 20min	- For the detection of EdU with Alexa Fluor® 647 azide use 633/635 nm excitation with a red emission filter (660/20 nm or similar).

Protocol tips
- Cells are labelled with EdU for 20min
Downstream tips
- For the detection of EdU with Alexa Fluor® 647 azide use 633/635 nm excitation with a red emission filter (660/20 nm or similar).

Publication protocol

For DNA-content analysis, trypsinized cells were centrifuged, and the pellets were washed in PBS. Cells were fixed in 4% paraformaldehyde in PBS for 15 min on ice and washed in PBS. Cells were then stained with Hoechst 33342 for 30 min. For analysis of labeled mitosis, the Click-iT EdU Pacific Blue Flow Cytometry Assay kit (Invitrogen) was used in combination with an M-phase marker, phospho-histone H3. Briefly, cells were incubated in growth medium containing 10 μM EdU (Invitrogen) for 20 min immediately after irradiation, and then washed in PBS. After the indicated time, cells were fixed in 4% paraformaldehyde in PBS for 15 min, and then stained using azide conjugated with Pacific Blue. Next, cells were incubated for 1 h on ice with primary monoclonal antibody against phospho-histone H3 (Ser10) (1:50; Cell Signaling, Danvers, MA). Following extensive washing, cells were incubated in goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 647 (1:500; Invitrogen) for 30 min on ice. After staining, all samples were washed in PBS. Finally, single-cell suspensions were passed through a nylon mesh. Each sample was analyzed on a FACS CantoII cytometer (Becton Dickinson, Franklin Lakes, NJ) using the FlowJo software (Tree Star, Ashland, OR, USA). At least 103 mitotic cells were analyzed for each time point. Duration of S phase was defined as the interval between 50% levels in the ascending and descending portions of the curve. Duration of G2 plus 1/2 M was determined from the interval between the 0% and 50% level on the ascending portion of the curve

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit below.

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