Fitc conjugated mouse anti human cd24

Panc‐1 Tet‐One, Tet‐MIST1, and Tet‐PTF1a cells were dosed with 1 μg·mL⁻¹ doxycycline and incubated at 5% CO₂, 37 °C. The medium was changed after 48 h, and cells were harvested at 72 h. Cells were pelleted, washed, and incubated with blocking antibody CD16/32 (Cat. 2.4G2, BD Pharmingen, Franklin Lakes, NJ, USA), washed twice with PBS, and then incubated with FITC‐conjugated mouse anti‐human CD24 (1 : 100, Cat. 560992; BD Biosciences) and Cy5‐conjugated CD44 (1 : 100, Cat. 553135; BD Biosciences) for 30 min at 4 °C. Cells were analyzed for coexpression of CD24 and CD44 antigens using the BD Fortessa Cell Analyzer in the Purdue University Bindley Biosciences Flow Cytometry Core. Gates were set using a no‐antibody control. Assays were performed with a minimum of three biological replicates.

Flow cytometry was performed with PE-conjugated mouse anti-human CD44 and FITC-conjugated mouse anti-human CD24 antibodies (both from BD Biosciences, San Diego, CA, USA) on a FACS Canto II instrument (BD Biosciences, San Diego, CA, USA), and data were analyzed with Cell Quest software (BD Biosciences).

Merck gamma secretase inhibitor (MK-0752) was provided by Dr. Max Wicha (University of Michigan) and was used for the in vitro and ex vivo studies. Gamma secretase inhibitor RO4929097 was kindly provided by Roche (Indianapolis, IN) and was used for the in vivo studies. PE-conjugated mouse anti-human CD44 and FITC-conjugated mouse anti-human CD24 antibodies were purchased from B.D. (Franklin Lakes, NJ). APC conjugated mouse anti-human ESA was purchased from Miltenyi Biotech and biotinylated mouse anti-mouse H2K was purchased from Southern Biotech. Hes1 antibody used for Western Blot was obtained from Dr. Xing Fan (University of Michigan) or purchased from MBL International (Woburn, MA). Notch1 and cleaved Notch1 antibodies were purchased from Cell Signaling Technology (Danvers, MA) and β-actin antibody was purchased from Sigma (St. Louis, MO). Matrigel was purchased from B.D. (Franklin Lakes, NJ). Hes1 shRNA clone V2LHS 249784 was purchased from Open Biosystems (Huntsville, AL). The delta/Serrate/Lag-2 (DSL) peptide (sequence CDDYYYGFGCNKFCRPR) was synthesized by the University of Michigan Protein Structure Facility.