Cell Surface ICAM-1 and GM1 Expression

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.