Pe conjugated cd24

Cell labeling was carried out by staining with antibodies in buffer composed of PBS supplemented with 1% BSA. Cells were first suspended and placed in a blocking solution on ice at a concentration of 1 × 10⁵ cells in a total volume of 0.5 mL for 30 min and then stained with conjugated antibodies for 45 min on ice in the dark. After centrifugation at 250× g for 5 min at 4 °C, cells were washed with cold staining buffer before being subjected to flow cytometry. The antibodies used in this study were PE-conjugated CD 24 (BD Bioscience, Franklin Lakes, NJ, USA) and FITC-conjugated CD29 (Biolegend, San Diego, CA, USA). Proper isotype controls were used for each cell labeling experiment. The main cellular population was gated on FSC/SSC, then doublets were removed and the remaining cells were analyzed for percentages of cells showing CD29+/CD24+, CD29−/CD24+, CD29+/CD24−, or CD29−/CD24−. Flow cytometry was performed on a Guava 6HT-2L flow cytometer (Luminex, Austin, TX, USA) and data analyzed using FlowJo, LLC software (Mac Version 10.8.0 Ashland, OR, USA).

We followed a previously described method [39 (link)]. A total of 1 × 10⁶ cells were incubated with FITC-conjugated CD44 and PE-conjugated CD24 (BD) and incubated at 4 °C for 20 min. The breast cancer cells were washed twice with 1XPBS and then analyzed by using a flow cytometer (Accuri C6, BD).

The pancreatic cancer cells in the logarithmic growth stage were collected and centrifuged. The concentration of cells was adjusted to 1×10⁶/ml.
For analysis of stemness markers, cells (1×10⁶) were labeled with PE‐conjugated CD24 (BD PharMingen™, cat. no. 555428) and APC‐conjugated anti‐CD44 (BD PharMingen™, cat. no. 559942), incubated at 4℃ in the dark for 30 min. Cells were then centrifuged and resuspended in PBS at a concentration of 1×10⁸/ml, and subsequently sorted on a flow cytometer (BD Accuri C6 Plus; BD Biosciences).
For apoptosis and necrosis analysis, cells were cultured for 12 h prior to being treated with gem for a further 24 h. For flow cytometry analysis, cells were detached and labeled using an Annexin V‐PE/7‐AAD Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd.; cat. no. KGA1017) according to the manufacturer's protocol. Apoptotic and necrotic cell subsets were quantified using flow cytometry. A total of 2×10⁴ cells were analyzed per sample. Annexin V‐PE⁻/7‐AAD⁻ cells were considered as viable, Annexin V‐PE⁺/7‐AAD⁻ cells were considered as early apoptotic, and Annexin V‐PE⁺/7‐AAD⁺ cells were considered as late apoptotic and necrotic cell populations.