Click it edu alexa fluor 488 kit

Cell cycle distribution was analyzed using EdU (added 45 min before harvest) to label cells in S phase and DAPI to label DNA content. D283 cells were treated with DMSO (0.1%), 7.5 μM THC or 5.5 μM CBD, in the presence of DMSO or 10 μM 4HPC. Time of harvest is indicated in the figures. Cells were stained using the Click-iT EdU AlexaFluor488 kit (Invitrogen). In addition, cells were stained with AlexaFluor647-conjugated cleaved PARP (CST #68975, 1:50) to identify apoptotic cells, and PE-conjugated phospho-histone H3 Ser10 (CST #5764, 1:50) to mark cells in mitosis. Samples were analyzed using an LSRFortessa X20 (BD, Franklin Lakes, NJ, USA) and results were visualized and quantified using FlowJo software. Data are pooled from two independent experiments and show the mean with standard deviation (SD).

Total cell number and viability was determined by trypan blue (Tb) exclusion or propidium iodide (PI). PI was added at 1 μg/ml 1 min before flow cytometry on a BD Biosciences FACS Canto Flow Cytometer (Universidad de Chile). For Tb, the number of viable cells was determined counting at least 100 cells per sample in triplicate. Cell proliferation rate was measured with the Click-iT® EdU Alexa Fluor® 488 kit (Invitrogen) according to manufacturer's directions. Samples were analyzed on an Olympus BX-51 fluorescence microscope.

Islets from three fetuses were used for each hormone study. At least 50 islets in each Petri dish were incubated with either T₃ (Sigma), insulin (Humulin‐R, Lilly, Indianapolis, IN, USA) or leptin (Abcam, Cambridge, UK) at three doses: 0.1, 1 and 10 ng ml⁻¹. The effects of T₄ were not assessed as T₃ is the active form of thyroid hormone. An additional dish of islets incubated with 10 ng ml⁻¹ IGF‐I (Sigma) was used as a positive control as IGF‐I is known to stimulate beta cell proliferation (Hogg et al. 1993). The islets were subjected to 95% O₂/5% CO₂ gas for 5 min in a humidity chamber before incubation at 37°C. After 24 h, the islets were transferred into fresh medium supplemented with hormone and 5‐ethynyl‐2′‐deoxyuride (EdU) at a final concentration of 10 μm. After a total incubation time of 48 h, the islets were fixed in 4% paraformaldehyde and frozen in optimum cutting temperature compound (Tissue Tek, Torrance, CA, USA). Islets were sectioned at −20°C to a thickness of 10 μm at 100 μm intervals and labelled for EdU‐positive cells using the Click‐iT EdU Alexa Fluor‐488 kit (Invitrogen, Grand Island, NY, USA).

The fraction of cells actively undergoing DNA synthesis was evaluated using 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays. OVCA420, UWB1.289, OVCAR3, or CAOV3, in some experiments with shRNA knockdown of SPINK1 as described, were seeded in 6-well plates at 1×10⁵ cells per well (OVCA420, UWB1.289, or CAOV3) or 3×10⁵ cells per well (OVCAR3). The next day media were changed to serum-free media and in some experiments cells were treated with varying concentrations of rSPINK1. After 24 hours, EdU incorporation was conducted using the Click-iT EdU Alexa Fluor 488 kit (Invitrogen). Following a 2 h interval of EdU incorporation, cells were fixed with 3.7% formaldehyde in phosphate buffered saline (PBS), permeabilized with 0.5% Triton X-100 in PBS, reacted with Alexa Fluor 488 azide, and nuclei were counterstained with Hoechst 33342, all according to manufacturer instructions. In some experiments, cells were subsequently blocked with 5% nonfat dry milk in PBS for 10 minutes at RT, incubated overnight at 4°C with 1:200 monoclonal anti β-tubulin clone TUB 2.1 (Sigma-Aldrich cat # T4026), and then stained with 1:500 goat anti mouse Alexa Flour 546 for 30 min at room temperature. Cells were rinsed with PBS and pictures were taken at 40× magnification for manual cell counting and calculation of proliferative indices.

Cell cycle analysis was carried out using the Click-iT^® EdU Alexa Fluor 488 Kit (Invitrogen, Thermo Scientific Inc., Waltham, MA), following the manufacturer’s instructions. Spheroids were exposed to EdU (5-ethynyl-2’-deoxyuridine, 10 μM) at 37 °C for 2 h. Thereafter, spheroids were trypsinized and the cells were FACS-analyzed. Dapi was used to stain total DNA and forward scatter to determine cell size.
Apoptosis was assessed by flow cytometry using the Annexin A5/7-AAD kit (Beckman Coulter, #IM3614). The metabolic capacity of cells was determined using CellTiter-Glo^® 3D (Promega, Madison, WI, #G9682) according to the manufacturer’s protocol. Luminescence was measured with a microplate reader (Tecan^®, Austria).

Cell proliferation was measured by tracking new DNA synthesis using Click‐iT^® EdU Alexa Fluor^® 488 Kit (Invitrogen). EdU was added to culture medium at 5 μM for 2 h or 2–4 days depending on experiment. Labelled cells were detected using the Hermes WiScan‐automated cell‐imaging system (IDEA Bio‐Medical Ltd. Rehovot, Israel) and analysed using MetaMorph and ImageJ software. For propidium iodine (PI) staining: cells were fixed in 70% ethanol, treated with RNAse and stained with PI (0.1 mg/ml), monitored by flow cytometry using BD FACSCalibur and analysed by CellQuest and FlowJo software. 5 μM CFSE was added to MDM according to CellTrace™ CFSE Cell Proliferation Kit manufacturer protocol (ThermoFisher, Waltham, MA, USA), and cells were left in culture to show potential cell division for an additional 6 days. CFSE labelling was conducted according to CellTrace™ CFSE Cell Proliferation Kit manufacturer protocol. Cells were monitored by flow cytometry using BD FACSCalibur and analysed by CellQuest and FlowJo software.

Cell proliferation assay was performed by plating cells in 60 mm tissue culture dishes. The cell numbers were counted every other day in triplicate for 12 days. 1×10⁴ cells were plated on gelatin-coated 60 mm tissue culture dishes, and the cells were counted the next day for day one. Cell were then fed every other day and counted on the days that they were not fed for 12 days. The rate of DNA synthesis was measured using Click-iT-EdU Alexa Fluor 488 kit (Invitrogen) as recommended by the supplier. The assay measures incorporation of EdU (5-ethynyl-2′-deoxyuridine), a nucleoside analogue of thymidine, during cell proliferation. TSP1+/+ and TSP1−/− choroidal EC (5×10⁵) were plated on 60 mm tissue culture and incubated with 10 µM EdU in culture medium for 3 h at 33°C. The DNA synthesis was analyzed by measuring incorporated EdU using FACSscan caliber flow cytometry (Becton-Dickinson).