Alexa fluor 647 conjugated cholera toxin b subunit

As previously described (Sasaki et al., 2015 (link)), cells were harvested with Accutase^® cell detachment solution (Merck Millipore, Billerica, MA, United States), and dissociated single cells were incubated with primary antibodies diluted in fluorescent activated cell sorting (FACS) buffer (0.5% [w/v] bovine serum albumin [BSA] and 0.1% [w/v] sodium azide in PBS) for 30 min on ice. After washing, the cell suspension was incubated with Alexa Fluor^® 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR, United States) and diluted in FACS buffer for 30 min on ice. For the detection of GM1 or c-series gangliosides, cells were incubated with Alexa Fluor^® 647-conjugated cholera toxin B subunit (Molecular Probes) or APC-conjugated anti-A2B5 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) diluted in FACS buffer for 30 min on ice, respectively. Cell sorting and analysis were performed using a FACSAria™ Cell Sorter. The following primary antibodies were used: anti-GM3 (NBT Laboratories Inc, Tokyo, Japan), anti-GM2 (TCI, Tokyo, Japan), anti-GD1a (TCI), anti-GT1b (TCI), anti-GD3 (Merck Millipore), anti-GD2 (TCI), and anti-GD1b (TCI). Mean fluorescence intensity was calculated by subtracting the intensity of the control.

FACS analysis of gangliosides and cell sorting in PDAC cells were performed as previously reported^{50 (link),51 (link)}. Cells were harvested and dissociated single cells were incubated on ice for 30 min with anti-GM2 antibody (TCI, Tokyo, Japan), anti-ganglioside GM3 (NBT Laboratories, Tokyo, Japan), anti-ganglioside GD1a (TCI), anti-ganglioside GD3 (Merck Millipore), anti-ganglioside GD2 (TCI), and anti-ganglioside GD1b (TCI) diluted in FACS buffer (0.5% [w/v] BSA and 0.1% [w/v] sodium azide in PBS). After washing, the cell suspension was incubated on ice for 30 min with Alexa Fluor® 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) diluted in FACS buffer. For ganglioside GM1 detection, cells were incubated with Alexa Fluor® 647-conjugated cholera toxin B subunit (Molecular Probes) diluted in FACS buffer for 30 min on ice. Cells were then washed and suspended in cell sort buffer (25 mM HEPES pH 7.0, 1 mM EDTA, 1% BSA in PBS). Cell sorting and analysis were performed using a FACSAria™ Cell Sorter (Becton Dickinson, Franklin Lakes, NJ, USA). Mean fluorescence intensities (MFIs) were calculated by subtracting the intensities of the controls.

As described in our previous reports [8 (link), 9 (link)], cells were harvested with the Accutase® cell detachment solution (Merck Millipore, Billerica, MA, USA) and dissociated single cells were incubated with FITC-conjugated ICAM-1 (BioLegend, San Diego, CA, USA) or FITC-isotype control (Becton Dickinson, Franklin Lakes, NJ, USA) diluted in FACS buffer (0.5% [w/v] BSA and 0.1% [w/v] sodium azide in PBS) for 30 min on ice. To detect GM1, cells were incubated with Alexa Fluor® 647-conjugated cholera toxin B subunit (Molecular Probes, Eugene, OR, USA) diluted in FACS buffer for 30 min on ice. After washing, cell sorting and analysis were performed using a FACSAria™ Cell Sorter (Becton Dickinson). Mean fluorescence intensities (MFIs) were calculated by subtracting the intensities of the controls.