Click it plus alexa fluor 647 flow cytometry assay kit

BRCA2^+/+ and BRCA2^−/− DLD1 cells treated for 48 h with 1 μM of cisplatin or 1 μM chlorambucil were incubated with 10 μM EdU for 1 h at 37°C. Cells were harvested by trypsinisation and processed for EdU staining using the Click‐iT Plus Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific). Cells were incubated with 20 μg/ml propidium iodide and 10 μg/ml RNase A (Sigma) in PBS. At least 10,000 cells were analysed by flow cytometry (FACSCalibur, BD Biosciences). Data were processed using FlowJo (FlowJo, LLC) software.

Cultures of PBMC at density of 2 X 10⁶ cells/ml in complete medium were stimulated for 72 h with PHA (5 ug/ml) in 96 well flat bottom plates (Corning Inc. Corning, NY). Cell proliferation was assessed by adding 5-ethynyl-2'-deoxyuridine (EdU) (ThermoFisher Scientific) at a concentration of 0.01 mM, which was added 24 h after the cells were initially activated with PHA. Non adherent cells were collected After 48h hours of additional culture, and EdU incorporation was measured using Click-iT™ Plus Alexa Fluor™ 647 Flow Cytometry Assay Kit (ThermoFisher Scientific), following manufacturer’s instructions. To analyze T cell proliferation, cells were stained with an anti-CD5 FITC conjugated antibody for canine T cells (rat anti dog clone YKIX322.3, ThermoFisher Scientific) or an FITC conjugated anti-CD3 antibody (mouse anti human clone UCHT1, ThermoFisher Scientific) for human T cells. Flow cytometry was performed as previously described^{18 (link)}. Cells were analyzed on a Beckman-Coulter Gallios flow cytometer. Cell analysis was done using FlowJo v9 software (BD Life Sciences, Franklin Lakes, NJ). Examples of FlowJo gating schemes for human and canine raw dot plots are shown in (Supplementary Fig. 1).