Alexa fluor 488 conjugated anti cd44 antibody

Panc-1 cells (8 × 10⁵) seeded in 6 cm plates were collected using a cell scraper with l × PBS containing 0.5% bovine serum albumin. The cells were washed twice with l × PBS and resuspended in 100 μl of Cell Staining Buffer (BioLegend). Subsequently, 2 μl of fluorescein isothiocyanate-conjugated anti-human CD133 antibody (130-113-673, Miltenyi Biotec) or Alexa Fluor 488–conjugated anti-CD44 antibody (103016, BioLegend) was added and incubated for 20 min at 4 °C in the dark. Cells were washed twice and resuspended in 750 μl of Cell Staining Buffer. Samples were analyzed using the EasyCyte Mini Flow Cytometer (Luminex) and FlowJo software (BD Bioscience, version 10, https://www.flowjo.com/).

PBS or rHSP90-treated HCT-115 or SW480 cells were trypsinized and collected, 1×10⁵ cells in 50 μl of ice-cold PBS plus 1% FBS were added with Alexa Fluor 488-conjugated anti-CD44 antibody or phycoerythrin-conjugated anti-CD326 antibody at the concentrations suggested by the manufacturer (BioLegend, San Diego, CA, USA). After 1-h incubation at room temperature and three times 20-min washes with PBS plus 1% FBS, the cells were resuspended in PBS plus 1% FBS and analyzed immediately by FACSCalibur flow cytometer (BD Biosciences).