Click ittm edu alexa fluor 488 imaging kit

The Click-iTTM EdU Alexa Fluor 488 Imaging kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was used to assess cell proliferation. Cells were visualized using an ImageXpress Micro XL automated digital imaging and High Content imaging and Analysis system (Molecular Devices LLC, San Jose, CA, USA).

A fasting, venous blood sample (approximately 50 ml) will be taken at rest. For visits 1 and 2, a further venous blood sample (approximately 100 ml total) will be taken after exercise testing. Blood samples will be centrifuged, separated within 30 min, and stored at − 80 °C. Fasting lipid and metabolic profiles, including C-reactive protein, will be measured at the Oxford Hospital Biochemistry using routine clinical quality validated assays. Additional circulating biomarkers will be quantified using standard commercially available assays [16 (link)]. Circulating endothelial colony forming cells (ECFCs) and peripheral blood mononuclear cells (PMBCs) will be isolated and cultured from peripheral blood as previously described [28 ]. Cells in culture will be followed from days 7–30 to assess ECFC colony formation. ECFCs will then be expanded to allow cell phenotype, clonogenic and functional assessments including cell proliferation (EdU incorporation) using the Click-iTTM EdU Alexa FluorTM 488 Imaging kit (Thermo Fisher Scientific), cell migration and vascular cord formation capacity assessed by the numbers of closed tubes and branching formed on Matrigel (Corning Matrigel Matrix). Remaining PBMCs and ECFCs will be frozen in 10% dimethyl sulfoxide (Sigma Aldrich) fetal bovine serum (Gibco, Thermo Fisher Scientific) and stored in liquid nitrogen.

The proliferation rate was assessed through the quantification of 5‐ethynyl‐2′‐deoxyuridine (EdU) cellular incorporation using the Click‐iTTM EdU Alexa FluorTM 488 Imaging kit (Thermo Fisher Scientific). There were 4.0×10⁴ ECFCs plated on collagen‐coated flasks, 1.86‐cm² surface area, maintained with complete EBM‐2 media, and grown for 24 hours. Similar culture conditions were used for all individuals. Cells were then incubated with 10 µmol/L EdU in complete EBM‐2 media for 4.5 hours at 37 °C, then fixed with 3.7% formaldehyde in PBS, permeabilized with 0.5% Triton X‐100 in PBS, and stained according to kit instructions. Assays were performed in triplicate and cell images obtained using a fluorescence microscope (Leica, Wetzlar, Germany) and a ×10 objective. The number of cells under proliferation and incorporating EdU into their DNA, stained positive for EdU (AlexaFluor 488), were counted in 3 to 5 full pictures per assay, as well as the total number of cells with nuclei stained with 4′,6‐diamidino‐2‐phenylindole. The percentage of proliferating cells (a measure of proliferation rate) was then calculated.