Calcein am dye

For growing PCa cells on 3D matrix, prechilled chamber slides were coated with a thin layer of Growth Factor reduced Matrigel matrix (BD Biosciences, Sane Jose, CA) maintained at 4°C. The cells were suspended in serum-free DMEM/F12 medium and added to matrigel (4 : 1 matrix to media) maintained on ice. From this mixture, a total of 50 μl (10⁴ cells/well) was transferred to the matrigel precoated chamber slide wells. The cells were grown for 7 days and the cell growth medium was replenished once every two days. After 7 days, C12-HSL (75 μmol/L) was added consecutively for 5 days with change in cell media and maintained at 37°C for the duration of the study. After the treatment, the cells were washed with 1x PBS and were imaged in an Olympus CK40 phase contrast inverted microscope to image the morphology of the cells. In separate experiments, Calcein AM dye (Ex/Em = 494/520 nm) (Biotium, Hayward, CA) was added to the cells after C12-HSL treatment and incubated for 2 h at 37°C to test the cell viability and cell membrane integrity. The cells were imaged using an Olympus CK40 fluorescent microscope. The corrected total cell fluorescence (CTCF) was calculated using ImageJ (NIH) using the following formula: CTCF = Integrated Density − (Area of selected cell × mean fluorescence of background readings) [26 (link)].

The 3D culture of Panc-1, Aspc-1, and HPDE cells was performed according to a model described earlier [19] (link). Briefly, the cells were trypsinized from a monolayer to single cell suspension and pelleted by centrifugation. Prechilled chamber slides were coated with a thin layer of Growth Factor Reduced BD Matrigel matrix (BD Biosciences, Sane Jose, and CA) maintained at 4°C. The cells were suspended in serum free (DMEM/F12) media and added to matrigel (4∶1 matrix to media) on ice. From this mixture a total of 60 µl (10⁴ cells/well) was added onto the matrigel-precoated chamber slides. After two weeks, O-DDHSL (200 µM) was added to cells in matrigel, and further incubated for 48 h at 37°C. Following incubation, Calcein AM dye (Ex/Em = 494/520 nm) (Biotium, Hayward, CA) was added to the cells and further incubated at 37°C for 2 h to check cell viability and membrane integrity. The cells were photographed using an Olympus CK40 fluorescent microscope.