Annexin 5 fitc and propodium iodide staining kit

The effect of cdPM treatment on cell viability, apoptosis and necrosis were determined using Annexin V-FITC and propodium iodide (PI) staining Kit (Invitrogen™, Eugene, OR) by flow cytometry. THP-1 cells were seeded into 12-well plates and treated with cdPM. After 24 hr of exposure, cells were washed with PBS, trypsanized to detach the cells, and re-suspended in Annexin V buffer according to manufacturer’s recommendations. A total of 100 μL of the cell suspension was mixed with 5 μL of FITC Annexin V and 1 μL of 100 μg/mL PI.
The mixture was incubated in the dark for 15 min at room temperature after which an additional 200 μL of Annexin V buffer was added. Samples were then placed on ice and analyzed by flow cytometry (FACSCanto BD Biosciences San Jose, CA) at emission wavelengths of 530 and 670 nm, respectively, within one hour. Assays were carried out in triplicate (33 μg/mL dose for 24 hr) with 10,000 cell events for each analysis. Population means were aligned via the use of single-color controls and compensation by the analytical software (Flowjo Version 10).

The cells were analyzed for viability, apoptosis, and necrosis using Annexin V-FITC and propodium iodide (PI) staining Kit (Invitrogen™, Eugene, OR, USA) by flow cytometry. After the removal of the basolateral media (saved for ELISA), 300 μL of TRYP-LE (Gibco, Thermo Fischer Scientific, MA, USA) solution was added to the apical side of the insert to detach the cells. After incubating for 3 min, 0.7 mL of warm DMEM media with FBS was added to the apical side. The cells were mixed using pipetting and finally transferred to 1.5 mL vials. The vials were centrifuged at 1200×g for 6 min. The supernatant was aspirated, and 300 μL of Annexin-V buffer was added to the pellet. The cells were resuspended and counted. A volume corresponding to 100K cells (~100 μL) was transferred to another set of 1.5 mL vials and mixed with 5 μL of FITC Annexin V and 1 μL of 100 μg/mL of PI. The mixture was incubated in the dark at room temperature for 15 min, and an additional 400 μL of Annexin-V buffer was added. Quickly, the cells were analyzed using flow cytometry (Beckman Coulter Cytoflex, Beckman Coulter, Life Sciences, IN, USA) at the emission wavelength of 525/40 and 690/50 nm. For each analysis, 30,000 cell events were recorded. Gating and analysis were performed using FlowJo 10 (OR, USA).