C12fdg

Murine MCs were harvested by peritoneal lavage as follows: naive mice were sacrificed by cervical dislocation and 5 mL of lavage buffer (2 mM EDTA, 0.25% BSA in PBS) was injected into the peritoneal cavity and incubated for 3 min prior to collection. MCs were identified within the harvested cell suspension as described in the flow cytometry section using a LSR Fortessa flow cytometer (BD Biosciences) and FlowJo software (TreeStar) and subsequent parameters were analyzed. To quantify MC senescence, cells were incubated in suspension in OptiMEM buffer (ThermoFisher Scientific) containing 100 nM of Bafilomycin (ThermoFisher Scientific) for 1 h at 37°C in order to increase the intracellular pH. The cell suspensions were then supplemented with 33 μM of C₁₂FDG (ThermoFischer Scientific) for 2 h prior to analysis of C₁₂FDG MFI. MC apoptosis was assessed as previously described (Asai et al., 2001 (link)). Briefly, peritoneal cells were stained with fluorescently labeled primary mAbs as described in the flow cytometry section and incubated with 500 μl of Annexin V binding buffer (140 mM NaCl, 2.5 mM CaCl₂ and 10 mM HEPES at pH 7.4) containing 5 μl of FITC-Annexin V (BD) and 5 μl of propidium iodide (Biolegend) for 15 min. Apoptotic cells were defined as Annexin-V⁺/propidium iodide^-. MC granularity was assessed using the flow cytometric side scatter profile.

To assess senescence status, we used a β-gal imaging method. In brief, cells were split into 12-well dishes (0.125 × 10⁶ cells per well) with a glass cover slide at the bottom of each well and allowed to settle for 24 hours. Cells were first pretreated with bafilomycin A1 (Selleckchem, S1413, 622.83 g/mol, 100 μM stock). Existing medium was aspirated, and then cells were washed with phosphate-buffered saline (PBS) and replaced with treated bafilomycin A1 media for 30 min at a final concentration of 100 nM. Following bafilomycin A1 pretreatment to normalize lysosome activity, C12FDG (Invitrogen, #D2893, 853.92 g/mol, 10 mM stock) was added directly to the existing media for 90 min at a final concentration of 10 μM. Note that because of light sensitivity, exchange was conducted in a dark environment. Following bafilomycin A1 and C12FDG treatments, medium was aspirated, and cells were washed with PBS three times, fixed with 4% paraformaldehyde/PBS (10 min), followed by two PBS washes, and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, #P36935) and mounted onto coverslips. Fixed cells were immediately imaged using a ZOE fluorescent cell imager (Bio-Rad). Percent cell positively was calculated using ImageJ with a background/negative threshold value. Any cells above this threshold were considered positive. All cells in each image frame were counted.

Measurement of SA-β-gal activity by FACS analysis was done as described elsewhere (36 (link)). In brief, 1x10⁶ single cells isolated from primary tumors, liver metastases and PC were seeded into one well of a 24-well-plate and incubated with 100 nM Bafilomycin A1 (Sigma-Aldrich) in serum-free medium at 37°C. Bafilomycin increases the pH in lysosomes to nearly neutral pH. After 1 hour the substrate 5-dodecanoylaminofluorescein-di-b-galactopyranoside (C₁₂-FDG, Fisher Scientific) was added at a final concentration of 50 µM. After 2 hours incubation at 37°C senescent cells have converted the non-fluorescent C₁₂-FDG to a fluorescent substrate. Finally, cells were washed in PBS and stained for viability with Zombie NIR (Bioloegend) and T cell markers as described in flow cytometry.