Annexin 5 fitc and pi apoptosis kit

Apoptosis detection was performed by using an Annexin V-FITC and PI apoptosis kit (eBioscience™, San Diego, CA, US). HL60 cells were plated at a 3 × 10⁵ cells/mL density onto a six well plate. After 24 h of incubation, the cells were treated with LEO at 0.2 mg/mL and with pure compounds at 77.1 µg/mL for terpinen-4-ol, linalool, and 1,8-cienole, and at 98.1 µg/mL for linalyl acetate. Cells grown in media containing an equivalent amount of DMSO served as the solvent control. After 24 h, the cells were stained with an Annexin V-FITC conjugate and propidium iodide, and the percentage of apoptotic, necrotic, and living cells was determined according to the protocol provided by the Annexin V-FITC and PI apoptosis kit. The cells’ emitted fluorescence was analyzed by flow cytometry (NovoCyte, ACEA Biosciences Inc, San Diego, CA, US) through the NovoExpress 1.3.0 software (ACEA Biosciences Inc, San Diego, CA, US), acquiring 1 × 10⁴ events per sample using the population plot “dot plot”, where each point corresponds to a single event with a specific fluorescence signal in reference to the axes; Annexin V-FITC green fluorescence in abscissa vs. PE red fluorescence in ordinate. The experiments were repeated three times.

Annexin V-FITC and PI apoptosis kit (eBioscience™) was used to perform the apoptosis detection. A total of 5 × 10⁵ SH-SY5Y cells /mL were seeded into a six well plate and, after 24 h of incubation, they were treated with HTyr-OL, HTyr and OA at their respective EC₅₀ values. Cells grown in media containing DMSO 0.001%, corresponding to the higher percentage applied, were used as solvent control (Ctrl), and cells treated with vinblastine were used as apoptotic control (VBL). Cells emitting fluorescence were analysed by flow cytometry after 24 h of treatment; the cells were stained with Annexin V-FITC conjugate and propidium iodide. The population plots (dot plot), obtained by acquiring 1 × 10⁴ events of each sample, were used to define the percentage of live cells (L), early apoptotic cells (EA), late apoptotic cells (LA), and necrotic cells (N), on the basis of the florescent signal emitted by each individual event in reference to the axes; Annexin V-FITC green fluorescence in abscissa, vs. PE red fluorescence in ordinate. The experiments were repeated three times.

For the surface marker staining, cells were stained according to the manufacturer's directions. The following antibodies were used: anti-F4/80 (eBioscience), anti-CD80 (eBioscience, Waltham, MA, USA), anti-CD86 (eBioscience), anti-MHCII (eBioscience). To detect LC3II in splenic macrophages, Flowcellect autophagy LC3 antibody-based assay kit (Merk Millipore) was used according to the manufacturer's directions. To assess TLR7, Notch1 expression in macrophages, spleen cells were then stained with anti-F4/80 and resuspended with fixation/permeabilization solution (eBioscience) then stained with anti-TLR7 (IMGENEX, CO, USA), anti-Noch1 (eBioscience), respectively. Similarly, fixed and permeabilized cells were stained with anti-Hes-1 (abcam) or anti-P62 (Merk Millipore), and then stained with Alexa Fluor 647 conjugated goat anti-rabbit IgG secondary antibody (abcam) and Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody (abcam), respectively. Annexin V-FITC and PI Apoptosis kit (eBiosciences) was used to examin the mortality of macrophages in vivo and in vitro. All of the flow cytometry data were aquired with the BD FACS Calibur cytometer and analyzed by FlowJo software.