Anti cd44 fitc and anti cd24 pe antibodies

After incubation with machilin D for 24 h, the cancer cells were harvested and dissociated using 1 × trypsin/EDTA. We used a previously described method [24 (link)]. A total of 1 × 10⁶ cells were cultured with anti-CD44-FITC and anti-CD24-PE antibodies (BD, San Jose, CA, USA) on ice for 30 min. The cancer cells were centrifuged and washed three times with a 1 × FACS buffer and analyzed by an Accuri C6 cytometer (BD, San Jose, CA, USA). The ALDH1 activity was assayed using an Aldefluor^TM assay kit (StemCell Technologies, Vancouver, BC, Canada). We used a previously described method [24 (link)]. The breast cancer cells were treated with machilin D (50 µM) for 24 h and reacted in ALDH assay buffer at 37 °C for 20 min. The ALDH-positive cells were examined by an Accuri C6 cytometer (BD, San Jose, CA, USA).

After incubating with tangeretin for 24 h, cancer cells were dissociated using 1× trypsin/EDTA. We used a previously described method [36 (link)]. In total, 1 × 10⁶ cells were cultured with anti-CD44-FITC and anti-CD24-PE antibodies (BD, San Jose, CA, USA) on ice for 30 min. The cancer cells were centrifuged and washed two times with 1× FACS buffer and analyzed on an Accuri C6 (BD, San Jose, CA, USA).