Alexa fluor 647 conjugated anti human ki 67

PBT cells (1×10⁶) were cultured in 300 µl of complete medium in 96-well plates. The cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum in a 5% CO₂ humid atmosphere at 37°C. PBT cells were cultured in the presence of medium alone, CSE or mAChR agonist/antagonist. The mAChRs were activated with muscarine (50 µM) and inhibited with atropine (100 µM) (Sigma-Aldrich or Tocris). In the proliferation assays, 1 µg/ml PHA and 50 ng/ml PMA (both from Sigma-Aldrich, St. Louis, MO) were added to the medium to stimulate T cell proliferation. After 5 days, the cells were harvested and then stained with APC-conjugated Annexin V and propidium iodide (Annexin V Apoptosis Detection Kit APC; eBioscience) at room temperature in the dark for 10 min. Finally, the proportion of apoptotic T cells was determined by flow cytometry. For T cell proliferation assays, intracellular staining with Alexa Fluor 647 conjugated anti-human Ki-67 (eBioscience) was performed.

Proliferation and apoptosis assays in vitro were performed as described in detail previously [12 (link)]. In brief, PBT cells were maintained in the presence of CSE, MR agonist muscarine (Mus, 50 μM, Sigma-Aldrich, St. Louis, MO, USA), and MR antagonist atropine (Atro, 100 μM, Sigma-Aldrich), either alone or in various combinations. In the proliferation assays, 1 μg/ml PHA and 50 ng/ml PMA (both from Sigma-Aldrich) were added to the medium to stimulate T cell proliferation. After 5 days, the cells were harvested and the proportions of proliferating and apoptotic T cells were determined by flow cytometry with Alexa Fluor^® 647-conjugated anti-human Ki-67 (eBioscience), allophycocyanin (APC)-conjugated Annexin V and propidium iodide (PI) (Annexin V Apoptosis Detection Kit APC; eBioscience).