Anti cd24 fitc and anti cd44 apc monoclonal antibodies

Apoptosis was measured by flow cytometry using the Apoptosis-PE Kit with Annexin V. Subpopulation of cancer stem cells was labeled as CD44+/CD24− using anti-CD24-FITC and anti-CD44-APC monoclonal antibodies (Becton Dickinson Biosciences, San Jose, CA, USA). Prostate cancer cells DU145 and PC3 (5 × 10⁴/mL) were seeded in 24-well plates for 24 h and then exposed to TRAIL in concentration of 100 ng/mL and/or one of the taxanes (paclitaxel, docetaxel or cabazitaxel) for 48 h. After this time the cells were harvested using trypsin and ethylenediaminetetracetic acid (EDTA), then washed twice with phosphate-buffered saline (PBS) solution and resuspended in 1× Binding Buffer (100 µL). The cell suspension was incubated with Annexin V-PE (5 µL), anti-CD24-FITC (20 µL) and anti-CD44-APC (20 µL) monoclonal antibodies for 20 min at 4 ℃ in the dark. After this time 400 µL of 1× Binding Buffer was added to each tube. The cells were analyzed by flow cytometry (LSR II, Becton Dickinson Biosciences, San Jose, CA, USA) within 1 h.

CD44+/CD24− subpopulation of cancer stem cells was labeled using anti-CD24-FITC and anti-CD44-APC monoclonal antibodies (Becton Dickinson Biosciences, San Jose, CA, USA). Prostate cancer cells DU145 and PC3 (5×10⁴/mL) were seeded in 24-well plates for 24 h and then exposed to TRAIL in concentration of 100 ng/mL and/or one of the taxanes (paclitaxel, docetaxel or cabazitaxel) for 48 h. After this time the cells were harvested using trypsin and ethylenediaminetetracetic acid (EDTA) and then washed twice with phosphate-buffered saline (PBS) solution and resuspended in PharmingenStain Buffer with BSA (100 µL) (Becton Dickinson Biosciences, San Jose, CA, USA). Then the cell suspension was incubated with anti-CD24-FITC (20 µL) and anti-CD44-APC (20 µL) monoclonal antibodies for 20 minutes in the dark. Cells in separate tubes treated with mouse IgG1 APC and IgG2a FITC monoclonal antibody constituted the isotype controls (Becton Dickinson Biosciences, San Jose, CA, USA). After incubation, 400 µL of PharmingenStain Buffer (BSA) was added to each tube. Finally, the cells were analyzed by flow cytometry (LSR II, Becton Dickinson Biosciences, San Jose, CA, USA) within 1 h.