Facscanto flow cytometry system

All cell samples were analyzed with a BD FACSCanto™ flow cytometry system (BD Bioscience, CA, USA), and statistical analysis was conducted in FlowJo software (FlowJo, OR, USA). The phenotype of T cells was assessed with fluorescently labeled antibodies specific for human CD3-PC5, CD4-fluorescein isothiocyanate (FITC) and CD8-FITC, which were obtained from BD Bioscience. Tumor surface antigen expression was detected with antibodies against human CD133-phycoerythrin (PE) (BioLegend, CA, USA) and GPC3-PE (Abcam, MA, USA); isotype control groups were stained with IgG1-PE (Abcam). The expression of GFP in T cells was evaluated FL1 channel to demonstrate the expression of CD133-CAR. The expression of GPC3-CAR was assessed by recombinant biotinylated protein L (Thermo Fisher Scientific) binding PE-conjugated streptavidin (PE-SA, BD Bioscience). All FACS-related cell samples were handled on ice and washed three times with 1 × PBS (Thermo Fisher Scientific) containing 1% FBS before staining the corresponding antibodies.

The intracellular ROS level was determined by using the DCFDA assay according to previous established protocol [24 (link)]. Human HPAF-II cells were cultured in a 6-well plate and treated with DHA (0, 50, 100, 150μM) and GEM (10μM) respectively, for 24hrs. Once cells were washed with PBS, they were later incubated with DCFDA at a final concentration of 10 μM in MEM FBS free medium for 30 mins at 37°C. Cells included as positive control were cultured with H₂O₂ at a concentration of 1mM. After incubation, cells were collected and resuspended in 500μl phosphate buffer saline (PBS), then analyzed by using BD FACSCanto flow cytometry system (BD Biosciences Inc., Franklin Lakes, NJ). The DCFDA fluorescence intensity represented the intracellular ROS levels in HPAF-II cells.

Cells were harvested by peritoneal lavage of thioglycollate-elicited female mice, aged 3 or 20 months, cultured overnight in complete DMEM to allow macrophage/monocyte cells to attach, washed with DPBS to remove non-adherent cells, and returned to culture to rest overnight as described above. To collect the adherent cells for analysis, plates were washed twice with DPBS to remove the residual culture medium and fresh ice-cold FACS buffer (1% bovine serum albumin and 0.05% sodium azide in DPBS) was added to the plate. Cells were gently removed using a cell lifter. The resulting cell suspension was pipetted up and down to achieve single cells and then passed through a 50-µM filter. Cells were stained with CD11b eFluor450 and F4/80 APC (eBiosciences) and 30,000 cells per sample were analyzed using a BD FACSCanto Flow Cytometry system.

For CSR from IgM to IgA, CH12F3-2A cells were stained with Dylite488-conjugated anti-mouse IgA (Abcam, Cambridge, UK) and APC-conjugated anti-mouse IgM (BD Pharmingen). For CSR from primary B cells, cells were stained with fluorochrome-conjugated anti-IgG1, anti-IgG₃, anti-IgE, anti-CD19, anti-B220, anti-CD43, anti-BP1, anti-CD24, anti-CD3, anti-IgD, anti-CD21, and anti-CD23 antibodies (all from BD Pharmingen). Cells were then processed on a BD FACSCanto flow cytometry system and the data were analyzed using FlowJo software (BD Pharmingen).

The lysosomal contents were assessed using LysoTracker Red (Invitrogen) following manufacturer’s instructions. After treatment, cells in fresh medium were stained with LysoTracker Red for 1 h followed by extensive washing and then transferred into tubes for quantification of lysosome fluorescence using a FACSCanto flow cytometry system (BD Bioscience). Data were analyzed by FlowJo software (Tree Star). For measurement of lysosome intensity using confocal image, the stained cells were fixed with 10% formalin solution followed by DAPI staining. Samples expressing fluorescence were identified by Olympus FV1200 confocal microscope.