Celltrace calcein red orange

For live cell imaging, saturated cultures were diluted 250× in fresh MB medium inside a μ-Slide 4- or 8-well slide (Ibidi) at time 0. To ensure oxygenation during the whole period of the experiment, the cover was removed. To maintain the temperature at 17 or 12°C, we used a P-Lab Tek (Pecon GmbH) Heating/Cooling system connected to a Lauda Ecoline E100 circulating water bath. To reduce light toxicity, we used a 495-nm Long Pass Filter (FGL495M- ThorLabs). For plasma membrane live staining (Fig 3B, S3 Video), FM4-64 (Invitrogen) at a final concentration of 10 μM from a 100× DMSO-diluted stock solution was added at time 0 unless indicated otherwise in figure legends. For cytoplasmic staining of cells in S3B Fig, cells were either stained with CellTrace CFSE Cell Proliferation Kit (Thermofisher) or CellTrace Calcein Red-Orange (Thermofisher).

Macrophages were treated with GO-BSA-FITC and, at the same time, labeled with CellTrace™ Calcein Red-Orange (ThermoFisher C34851, Waltham, MA, USA) at the final concentration of 500 nM for 30 min. After treatment, cells were washed three times with PBS, spotted on a glass slide, and analyzed with a LSM510 confocal microscope (Zeiss, Oberkochen, Germany).

Live cells were plated in 6-well plates and stained with Calcein (CellTrace^™ Calcein Red-Orange, ThermoFisher Scientific, UK) and Hoechst (ThermoFisher Scientific, UK) for cytoplasmic and nuclear high-content imaging (Operetta, Perkin-Elmer, UK). Fluorescence was assessed based on Atto647. The Operetta’s Harmony software was used for image analyses. Imaging was based on a randomized field analysis methodology that covered each of the studied wells.

Human umbilical vein endothelial cells (HUVECs) were obtained from Cell Applications (San Diego, CA). Tube formation assays were performed according to previously published protocols^{79 (link)}. Briefly, HUVECs were seeded at 2 × 10⁵ cells per 75 cm² in T75 culture flasks and expanded in Human EC Growth Medium (Cell Applications) until 75% confluence was reached. After washing off growth media, cells were serum starved one day before the assay by incubation in Human EC Basal Medium (Cell Applications) for 24 h. Calcein AM (green; ThermoFisher) was added to cultures at a concentration of 2 µg/ml to stain cells and assess cell viability. Cells were incubated with Calcein AM in the dark at 37 °C and 5% CO₂ for 30 min, then the stain was washed off using PBS. Assays were performed in 24-well plates coated with Geltrex LDEV (lactose dehydrogenase elevating virus)-Free Reduced Growth Factor Basement Membrane Matrix (ThermoFisher) at 75 µl/cm² according to the manufacturer’s recommendations. Cells were plated at a concentration of 3.5 × 10⁴ in 200 µl Human EC Basal Medium (Cell Applications) per well. For co-culture assays, DCs were stained using CellTrace Calcein Red-Orange (ThermoFisher) and added to the EC cultures at a 1:1 ratio. Assays were run for 12 h and then fixed using 4% paraformaldehyde and imaged using a Zeiss LSM 880 confocal laser scanning microscope.