Dead cell apoptosis kit with annexin 5 fitc

After 24 h of treatment with gemcitabine at IC₅₀ cell lines (2 nM in HAP1 cells and 1 nM in MEL202 cells) or cytarabine (0.1 µM in HAP1 cells and 0.2 µM in MEL202 cells), the supernatant was recovered, and adherent cells were harvested with trypsin and counted. The Dead Cell Apoptosis Kit with Annexin V FITC, propidium iodide (#V13242, Thermo Fischer Scientific), and flow cytometer ZE5 Cell Analyzer (BioRad) were used to evaluate the apoptosis rate of cells. The data analysis was done with FlowJo software 10.7.2 version (Supplementary Fig. 4).

Cells were seeded in collagen gels and allowed to grow for 10 days with or without DDR1i (2 µM). For EdU proliferation assay, EdU was added on day 10 and gels were allowed to grow for two more days and fixed for analysis on day 12. EdU fluorescence detection was performed according to the manufacturer’s protocol (C10640, Thermo Fisher). The percentage of EdU⁺ cells per organoid was analyzed in 10 organoids of each group. For cell cycle analysis, cells were harvested on day 10 and labeled with DRAQ5 membrane permeable dye (65-0880-96, Invitrogen) at a concentration of 5 µM and analyzed using a flow cytometer (BD LSRII at the Tufts Flow Cytometry Core). Analysis of cell cycle phases was performed on FlowJo software v10.7 using the Dean-Jett-Fox model. For dye dilution analysis, CellTrace CFSE cell proliferation kit for flow cytometry was used (C34570, Thermo Fisher) according to manufacturer protocol. Cells were labeled with 5 µM CFSE immediately before embedding in collagen gels, and isolated and analyzed by flow-cytometry (as above) after 3 days in culture. For Annexin V assay, Dead Cell Apoptosis Kit with Annexin V FITC was used (V13242, Thermo Fisher) according to manufacturer protocol. For cell cycle, dye dilution and annexin V analyses, 3 samples were individually cultured, treated, isolated and analyzed in each treatment group.

SK-MEL-05 and SK-MEL-147 cells were transduced with the AdRGD-PG-hIFNβ and AdRGD-CMV-LacZ (used in place of the eGFP-encoding vector in order to avoid interference with FITC) vectors using an MOI of 100, and collected after 24, 48 and 72 h incubation. Dead Cell Apoptosis kit with annexin V FITC (Thermo Fisher Scientific, # V13242) and propidium iodide were used to stain these cells following the manufacturer’s recommendations. Briefly, after washing in 1 × PBS, the cells were suspended in Annexin Binding Buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl₂, pH 7.4) and labeled with Annexin V and PI (10 mg / ml) for 10 min at room temperature, protected from light exposure. Cytometry and the resulting analyses were performed using the Attune instrument and NxT Flow Cytometry Software (Thermo Fisher Scientific).