Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit 1

The procedure of Hoechst 33258 staining was performed to evaluate apoptotic cells morphologically. For this, the untreated and treated neuron-like cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) at 4 °C for 20 min. Then, the cells were washed twice with PBS and stained with fluorescent Hoechst 33258 dye (1 mM, Sigma-Aldrich^®). Finally, cells were analyzed and photographed under the fluorescence microscopy (Leica^® DM IL LED) [20 (link)].
The rates of viable, apoptotic, and necrotic cells were also determined by the Annexin V-Fluorescein isothiocyanate (FITC) apoptosis detection kit I (BD Pharmingen^®). After incubation, 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI, 50 µg/ml) were added to the cultures and incubated in the dark for 10 min. Then, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline at 4 °C for 20 min and were analyzed by a flow cytometer (CyFlow Cube 6, Partec^®). Four cell populations types were determined with the Annexin V-FITC kit: (1) viable cells which were annexin negative and PI negative, (2) early apoptotic cells which were annexin positive and PI negative, (3) late apoptotic/necrotic cells which were annexin positive and PI positive, and (4) necrotic cells which were annexin negative and PI positive [21 (link),22 (link)].

Quantitative analysis of the apoptotic population was performed using the Annexin V‐fluorescein isothiocyanate (FITC) apoptosis detection kit I (BD Biosciences). After the miltefosine treatment or siRNA transfection, cells were collected and washed twice with cold PBS. The cells were resuspended in binding buffer (1 × 10⁶ cells/mL), and 100 μL of suspension was transferred to a tube and mixed with 5 μL of FITC Annexin V and propidium iodide (PI). Then, the mixture was incubated at room temperature for 15 min in the dark. After incubation, 400 μL of 1× binding buffer was added and then analysed by flow cytometry (BD AccuriTM C6, BD Biosciences)

The natural product viriditoxin was isolated from the marine-derived fungus P. variotii, as previously reported [8 ]. Briefly, P. variotii was cultured in a medium containing glucose (20 g/L), malt extract (20 g/L), peptone (1 g/L), and sea salt (26 g/L) at 30 °C on a shaker incubator (155 rpm) for 21 days, in a total volume of 22 L. The culture medium and mycelia were extracted with ethyl acetate (EtOAc). The EtOAc extract was partitioned into aqueous methanol (MeOH) and n-hexane; a yellow precipitate appeared at the interphase of MeOH and n-hexane layers. The yellow precipitate was filtered and identified as viriditoxin by proton nuclear magnetic resonance (¹H-NMR; 400 MHz). Paclitaxel and colchicine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against β-tubulin (sc-58886) and bovine anti-mouse IgG-horseradish peroxidase (HRP) (sc-2371) secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor^® 488-conjugated anti-mouse IgG secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit I was purchased from BD Biosciences (San Diego, CA, USA). All other chemicals were purchased from Sigma-Aldrich.

The cells were detached with trypsin and collected into a 15‐mL centrifuge tube. The cells were centrifuged at 800 g, after which the precipitation was washed twice with PBS. The cells were subsequently resuspended with 500 μL binding buffer based on the protocols of the Annexin V‐fluorescein isothiocyanate (FITC) Apoptosis Detection Kit I (556547, BD Biosciences). The cells were then permitted to react with 5 µL Annexin V‐FITC and 5 µL propidium iodide (PI) for 15 minutes under dark conditions. Finally, a flow cytometer was employed to detect cell apoptosis.

Hydrogen peroxide (H₂O₂) was bought from MP Biomedicals, USA. Keratinocyte-SFM medium, RPMI 1640 medium, penicillin, streptomycin, fetal bovine serum and l-glutamine were purchased from GIBCO, Invitrogen, USA. Annexin V-Fluorescein isothiocyanate (FITC) Apoptosis Detection Kit I (BD Pharmingen™) and Flow Cytometry Mitochondrial Membrane Potential Detection Kit were bought from BD™ MitoScreen, Becton–Dickinson Biosciences, USA. Camptothecin (CPT) was purchased from Santa Cruz Biotechnology, CA, USA. Ammonium acetate was bought from Merck, Germany. Chloroform was bought from R&M Chemicals, UK. Phenol and Sodium dodecyl sulfate (SDS) were procured from Amresco, USA. Isoamyl alchohol was purchased from Fluka, Switzerland. Phusion High-Fidelity DNA Polymerase was procured from Finnzymes, Finland. PCR primers were from First Base Laboratories. QIAquick Gel Extraction Kit and QIAquick Nucleotide Removal Kit were bought from QIAGEN, Germany. DNA Polymerase I Large (Klenow) Fragment, restriction enzymes and T4 DNA Ligase were obtained from New England Biolabs (NEB), USA. dNTP mix was purchased from Promega, USA.