Apc anti human cd44 antibody

To evaluate the changes of CD44 in MCF-7 after incubation with ADR/Exo, 1 × 10⁶ MCF-7 cells were cultured in six-well plates with different concentration (1 × 10⁵, 1 × 10⁶, 1 × 10⁷, 1 × 10⁸, and 1 × 10⁹ particles/mL incubation for 7 days) and different time (1 × 10⁸ particles/mL ADR/Exo harvested at days 0, 1, 3, 5, and 7) continuous passage cultures and added every other day. Cells were respectively harvested and stained with allophycocyanin (APC)-anti-human CD44 antibody at room temperature for 30 min (BioLegend, San Diego, CA, USA, CN. 338806, 4 μL of antibody in 100 μL of 1× PBS). APC-labeled IgG1κisotype controls (BioLegend, San Diego, CA, USA, CN. 3400119) were used as negative controls. CD44 expression in MCF-7, MCF-7/ADR, and MCF-7+ADR/Exo cell lines was assayed by flow cytometry analysis. According to the best time and concentration obtained, we further examined the DOX sensitivity of MCF-7 after 7 days of incubation with 1 × 10⁸ particles/mL ADR/Exo by Cell Counting Kit-8 (CCK-8).

For cell surface marker staining and flow cytometric analysis, MCF-7 or BT-474 cells were resuspended in PBS containing 1% FBS and incubated with PE anti-human CD24 antibody (1:20, clone: ML5, Cat# 311106, BioLegend) and APC anti-human CD44 antibody (1:20, clone: ML5BJ18, Cat# 338806, BioLegend) for 30 min at 4 °C. Afterwards, cells were washed with PBS. ALDH1 activity was measured using the ALDEFLUOR kit (Cat# 01700, Stem Cell Technologies) according to the manufacturer’s instructions. Specimens were subsequently analyzed by a CytoFlex Flow cytometer (Beckman Coulter). Data were analyzed using FlowJo software (version 10, Treestar).