Calcein am

Conditioned medium was collected from cultured GC cell groups. Before coating, the HLECs, pipette tips, Ibidi angiogenesis slides (Ibidi, German), and Matrigel (Corning, NY) were all precooled at 4 °C overnight. 5×10³ HLECs were seeded together with conditioned medium in Ibidi angiogenesis slides pre‐coated with Matrigel. Six hours later, the capillary‐like structures formed by HLECs were stained by Calcein‐AM (Biolegend, CA) and visualized via fluorescence microscope (DM4500B, Leica, German). Image J was used to measure the length of tubes.

Cell viability of prASCs was determined by by two viability assays, a photometric assay using 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT), as described previously [29 (link)], and a fluorescent-based assay using calcein-acetoxymethyl (calcein-AM, Biolegend, San Diego, USA), to determine any possible cytotoxic effects during the stimulations. In brief, 5000 cells/well were seeded in 96-well plates. We measured each stimulation in quintuplicate for each biological replicate. One day after seeding, the prASCs were stimulated for 96 h as described above. The XTT reagent was then added to the wells, as described by the manufacturer (AppliChem, Darmstadt, Germany), and incubated at 37 °C for 4 h. Absorbance was measured in an Apollo LB911 microplate reader (Berthold, Bad Wildbad, Germany) at 492 vs. 650 nm. Data are expressed as arbitrary units and calculated as a percent in relation to the control. For the fluorescence assay, cells were washed and calcein-AM (1 µM) was added incubated at 37 °C for 30 min. Then, fluorescence was measured immediately using a fluorescence reader (BMG Fluostar, Ortenberg, Germany) with excitation and emission wavelengths of 485 and 515 nm. Cells incubated in buffer without calcein-AM were used as background controls. Data are expressed as arbitrary fluorescence units.

Monocytes were sorted from the blood of Py8119 tumor-bearing mice and labeled with Calcein AM (Biolegend) following the manufacturer’s instructions. Labeled monocytes were plated in a 96 well plate on a monolayer of E2 endothelial cell line (20 (link)) and incubated for 1 h at 37°C. For activation of endothelial cells, 100 ng/ml mouse recombinant IL1β (R&D Systems) was added 5 hours prior to the addition of monocytes. After 1 h adhesion, the fluorescence signal at the bottom of the plate was measured at 520 nm (total fluorescence). The plate was then gently washed five times with prewarmed phenol red-free RPMI (Gibco) using a multichannel pipette. Fluorescence signal at the bottom of the plate was measured again at 520 nm (adherent fluorescence). The percentage of adherent monocytes was calculated as the percentage of adherent fluorescence signal compared to the total fluorescence signal.

Calcein AM (Dojindo Molecular Technologies, Inc.) and Annexin V-Alexa Fluor® 647 (BioLegend) dyes were used to stain viable and apoptotic cells, respectively. Hoechst 33258 (Dojindo Molecular Technologies, Inc.) was used to stain the nuclei. The staining solution was prepared by mixing 10 µL Hoechst 33258 (1 mg mL⁻¹ stock concentration), 10 µL Calcein AM (1 mg mL⁻¹ stock concentration), 10 µL Annexin V (50 µg mL⁻¹ stock concentration), 500 µL Annexin V binding buffer (BioLegend), and 500 µL DMEM. The wash buffer comprised 500 µL of Annexin V binding buffer and 500 µL of DMEM. After treatment, the cells were washed twice with fresh DMEM, and 10 µL of staining solution was introduced into a cell culture chamber using a pipette via the adjacent inlet. The cells were then incubated at 37 °C for 30 min. Excess staining solution was removed by washing with 30 µL of wash buffer three times.

In viable cells, Calcein-AM (calcein-acetoxymethyl) enters and becomes fluorescent upon hydrolysis via esterase enzyme. Its fluorescence is quenched by intracellular iron content [33 (link), 34 (link)]. Cells were incubated with 100 nM Calcein-AM (BioLegend) for 30 min at 37 °C in PBS. After Calcein-AM loading, the cells were trypsinized, washed and, re-suspended in PBS without Calcein-AM. They were then plated in 96-cell plates. Fluorescence was monitored using multifunctional enzyme mark at λex 488 nm and λem 518 nm (Thermo, USA) where a low Calcein-AM fluorescence intensity indicates high intracellular free iron level.

Hanging drops of melanoma cells (250 cells/ 25 μl-drop) in standard growth medium were cultivated for 7 days to induce spheroid formation. Ten spheroids were embedded in 1 ml medium with 1.2 mg/mL collagen I (Corning) in a 12-well plate. Light microscopic images were taken daily from day 0 to day 6. ImageJ software was used to quantify the spheroid diameter and size. At day 7, spheroids were stained with 8 µg/mL propidium iodide for cell death (Sigma Aldrich) and 2 µM calcein-AM (Biolegend) for viability and analyzed with an Axioplan 200 fluorescence microscope (Zeiss).