Annexin 5 propidium iodide

The different phenotypes of macrophages were examined using flow cytometric direct immunofluorescence. CD206 was used as a pan-macrophage marker, and iNOS and CD206 were used as specific markers of M1 and M2 macrophages, respectively [14 (link),15 (link)]. The cells recovered from the blood were rinsed in PBS and incubated for 30 min on ice with the following specific antibodies (all from eBioscience, San Diego, CA): anti-mouse CD68-FITC, anti-mouse iNOS-PE, anti-mouse CD206-FITC, and the isotype control. After fixing in 2% paraformaldehyde, flow cytometry analysis was performed. Intracellular ROS levels were measured by the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, China). Briefly, the cells were treated with NAC 2 h before treatment with geridonin and then stained with 10 μM DCHF-DA at 37°C for 30 min. After washing, the cells were assayed by BD LSR II flow cytometry. The PMECs and AT-II cells from each group were detached with 0.25% trypsin, and then were stained with annexin V/propidium iodide (PI) according to the kit manufacturer’s instructions (Beyotime, Beijing, China). The percentages of cells undergoing apoptosis were analyzed by flow cytometry immediately after staining.

The H9c2 cardiomyocytes were processed with Annexin V/propidium iodide (PI) staining according to the manufacturer’s instructions (Beyotime, Nantong, China). The samples were analyzed by a FACSCalibur flow cytometry (Becton Dickinson, Franklin Lakes, NJ).

To evaluate the anti-NNV activity of the drug delivery system in vitro, toxicity of SWCNTs-P-A and SWCNTs-P-A-Nb to SSN-1 cells was first checked by trypan blue staining test (44 (link)). SSN-1 cells were grown to a monolayer in 12-well plates and infected with 10² TCID₅₀ NNV. Following infection for 2 h, the cells were, respectively, treated with amantadine, SWCNTs-P-A, and SWCNTs-P-A-Nb with the same amantadine concentrations (3.125, 12.5, and 50 mg/L). Following treatment for 48 h, the relative NNV contents in SSN-1 cells were analyzed by reverse transcriptase quantitative PCR (RT-qPCR) and Western blotting. Moreover, apoptosis analysis was carried out based on Annexin V/propidium iodide (PI) staining (Beyotime Biotech, Nantong, China). Briefly, SSN-1 cells were grown to a monolayer in 6-well plates and treated as described above. Approximately 2 × 10⁵ cells were collected and stained with annexin V-FITC (5 μL) and PI (5 μL) following the manufacturer’s instructions. After staining, flow cytometry analysis was conducted immediately. FITC fluorescence (FL1) and PI fluorescence (FL2) of each cell were quantitated using the Cell Quest Pro software. The annexin V-FITC⁻/PI⁻, annexin V-FITC⁺/PI⁻, and annexin V-FITC⁺/PI⁺ populations were regarded as normal cells, early apoptosis, and late apoptosis, respectively.