Annexin 5 phycoerythrin

Monocytes were washed in 1× PBS and incubated in blocking solution consisting of FACS buffer (145 mM NaCl, 8.45 mM Na₂HPO₄, 1.83 mM NaH₂PO₄, and 0.1% NaN₃), 5% BSA, and human FcR binding inhibitor (eBioscience, San Diego, CA, USA) for 20 min on ice. After blocking, cells were stained by adding APC anti-human CD14, Alexa Fluor 488 anti-human CD16, PE/Cy7 anti-human CD163, Brilliant Violet 605 anti-human CD86 (BioLegend, San Diego, CA, USA), and APC-R700 anti-human CD80 antibodies (BD Biosciences, San Jose, CA, USA) or isotype controls on ice. Cells were then washed in 1× PBS and analyzed by flow cytometry using an LSRFortessa cell analyzer and BD FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA).
For viability studies, monocytes were washed after incubation with the APC anti-human CD14 antibody as described above. Cells were then stained with phycoerythrin-annexin V (BD Pharmingen, Franklin Lakes, NJ, USA) and either Sytox Blue dead cell stain (Life Technologies, Carlsbad, CA, USA) or propidium iodide (PI) dead cell stain (Thermo Fisher Scientific, Waltham, MA, USA) to detect dead and dying cells. After staining, cells were analyzed by flow cytometry as described above. Double negative cells represent live cells, whereas double positive and single positive cells represent dead and/or dying cells.

After drug treatment for 48 h, the 231 and 231/PTX cells were collected and suspended in binding buffer and then stained with Annexin V-Phycoerythrin (BD Biosciences) for 15 min at room temperature in the dark. Subsequently, the cells were analyzed by flow cytometry using Calibur (BD Biosciences) within 1 h.

Next, to find out the causes of the anti-proliferative effect of the PEF-III, an apoptosis-indicating experiment, annexin V-phycoerythrin and propidium iodide (PI)- staining, was performed. First, the MCF-7 cells (1 × 10⁶) were plated in a 10-cm dish and incubated at 37 °C and 5% CO₂ for 24 h. The cells were then treated with PEF-III and incubated for 2 days, after which they were washed in PBS twice, trypsinized, and resuspended in 100 μL of annexin-binding buffer (BD Company, Franklin Lakes, NJ, USA). After that, the resuspended cells were stained with annexin V-phycoerythrin (BD Company) and PI (BD Company) at a ratio of 1:10 and subjected to flow cytometry. FlowJo software (Tree Star, Ashland, OR, USA) was used for the apoptosis analysis.

Annexin V-Phycoerythrin (PE) and 7-Amino-Actinomycin (7-AAD) double staining method (BD Bioscience, Franklin Lakes, NJ, United States) was adopted to detect apoptosis. HL-7702 cells (Human normal liver cell line-7702) were cultured in a 6-well plate at a density of 1 × 10⁶ cells per well. After treatment, the cells were suspended in binding buffer, and then stained with PE and 7-AAD reagents for 15 min in the dark. The samples were analyzed using the BD FACS Calibur flow cytometer, and the data was analyzed using FlowJo software.

After treatment of the cells with cisplatin (48 hours) or radiation (72 hours), the floating and adherent cells were harvested, washed with PBS, and stained with Annexin-V-phycoerythrin (or Annexin-V-FITC) and 7-AAD (BD Biosciences, NJ) for 15 minutes at room temperature. The cells were then subjected to flow cytometry analysis (Beckman Coulter, Cytomics FC500, CA).