Fitc conjugated anti cd44

The expression of CD44 and absence of CD24 (CD44⁺/CD24⁻) is characteristic of breast CSCs. To evaluate these, Hs578T and Hs578Ts(i)₈ cell variants were seeded at 1 × 10⁵ cells in a 6-well plate and allowed to attach overnight. They were subsequently trypsinised, blocked with 10% FCS in PBS and stained with APC-conjugated anti-CD24 (1:100) (eBioscience, San Diego, California, USA) and FITC-conjugated anti-CD44 (1:400) (eBioscience) for 30 min at 4 °C. Staining was assessed in a FACSCanto II flow cytometer, followed by analysis using BD FACSDiva software. To assess the effects of 2-DG on the CSC population Hs578T and Hs578Ts(i)₈ cell variants were seeded at 1 × 10⁵ cells in a 6-well plate and allowed to attach overnight. Cells were treated with 2-DG (final concentration 15 mM) for 24 hours. They were subsequently trypsinised, blocked with 10% FCS in PBS and stained with APC-conjugated anti-CD24 (1:100) (eBioscience, San Diego, California, USA) and FITC-conjugated anti-CD44 (1:400) (eBioscience) for 30 min at 4 °C. Staining was assessed in a FACSCanto II flow cytometer, followed by analysis using BD FACSDiva software.

Adherent cells were detached with 0.05% Trypsin (Corning, Manassas, VA), washed and re-suspended in phosphate buffered saline (PBS). Tumors were mechanically and enzymatically disaggregated into single-cell suspensions, as in [35 (link)]. 1×10⁶ cells were incubated with PE-conjugated anti-CD133 and/or FITC-conjugated anti-CD44 (eBioscience, San Diego, CA; 12-1338-42 and 11-0441-82). Labeled cells were analyzed by LSR Fortessa Cell Analyzer (BD Bioscience). Data was analyzed with Flowjo software. At least three samples were analyzed in triplicate and a normal distribution was observed for biological replicates.