Pe conjugated ki 67

For the proliferation assay, naïve CD4⁺ T cells were purified by MACS beads (MiltenyiBiotec) and were labeled with CFSE (eBioscience) at 37°C water bath for 15 min. Labelled cells were stimulated in 96 well culture plates with plate bound anti-CD3 (5 μg/mL) and anti-CD-28 (1 μg/mL) in the presence of DMSO or BJ-3105 in Th1 and Th17 differentiation conditions: Th0 (anti-CD3/CD28 with no cytokines), Th1 (IL-12, 10 ng/mL plus anti-IL-4, 5 μg/mL), Th17 (IL-6, 10 ng/mL, TGF-β, 1 ng/ml plus anti-INF-γ and anti-IL-4, each at 5 μg/mL). After 72 h of culture, cell proliferation was assessed by CFSE dilution, using flow cytometry. For 5-bromo-2'-deoxyuridine (BrdU) labeling, naive CD4⁺ T cells from spleens and lymph nodes were isolated by immunomagnetic positive selection from 6–10 weeks C57BL/6 mice. The cells were cultured under Th1 differentiation condition with BrdU (10 μM). After 72 h of culture, cells were stained with APC-conjugated BrdU as per manufacturer’s protocols (BD Biosciences) in BrdU kit and cell proliferation was assessed by using flow cytometry. For Ki-67 detection, naive CD4⁺ T cells were isolated and cultured under Th1 differentiation condition and after 72 h of culture; cells were stained with PE-conjugated Ki-67 (Biolegend). Cell proliferation was assessed by using flow cytometry.

ECFCs proliferation was assessed through % expression of cell nuclear antigen Ki67 by flowcytometry. We investigated Ki67 expression in ECFCs of both CAD and control subjects at passage 3. Briefly, 1 × 10⁵ cells/well were seeded on a 6 well plate and incubated for 72 h at 37 °C, 5% CO₂. After incubation, cells were harvested and permeabilized by treating with pre-chilled 70% ethanol and incubated for 1 h at − 20 °C. Thereafter, cells were resuspended into staining buffer and stained with PE conjugated Ki67 (Cat. 350,503, BioLegend) antibody and incubated for 1 h at room temperature. Cells were acquired in FACS LSR-II (Becton Dickinson). The unstained cell sample was used as negative control and Ki67-PE signal in logarithmic mode was recorded and data expressed as % expression of Ki67 in histogram.