Pe conjugated anti cd133

CD133 is a marker of colorectal cancer stem cells (CSCs) (Dalerba et al. 2007 ). To assess the prevalence of CD133-positive cells in the population, cells were treated with 5FU, OXA or co-treatment for 48 h, followed by medium changing and culturing in DFM. The percentage of CSCs was analyzed after 15d at this condition. Briefly, cells were dissociated and washed once with PBS 1x containing EDTA 2 mM and 0.5% of BSA for 1h. After this, cells were incubated with PE-conjugated anti-CD133 (Miltenyi Biotec, Germany) 1:100 (v/v), for 10 min. Finally, cells were washed once with incubation buffer, resuspended, and analyzed by flow cytometry.

For flow cytometry, single-cell suspensions of A549 cells were incubated with the appropriate dilution of an isotype control or specific antibody. The antibodies used were PE-conjugated anti-CD133 or APC-conjugated anti-CD133 from Miltenyi Biotec (Bergisch Gladbach, Germany). After a 45-min incubation with primary antibodies, the cells were washed before analysis and sorting using a BD LSRII Flow Cytometer (BD Biosciences, San Jose, CA, USA). Sorted CD133 + and CD133 -A549 cells were cultured in DMEM supplemented with 10% fetal bovine serum (Biological Industries).
For the annexin V/propidium iodide (PI) assays, cells were stained with 10 µL annexin V-FITC and 10 µL PI (5 μg/mL) and stirred slowly for even mixing. Cells were placed in a dark room at room temperature for approximately 30 min and then 300 µL binding buffer were added (10 mM HEPES, pH 7.4, 140 mM NaOH, 2.5 mM CaCl 2 ). The cells were analyzed via flow cytometry and early apoptotic (annexin V-positive, PI-negative), late apoptotic (annexin V-positive and PIpositive), and dead (annexin V-negative and PI-positive) cells were counted to determine apoptosis and cell death.