Fitc annexin apoptosis detection kit 2

To confirm the ability of rhANXA5 to detect cells undergoing apoptosis, we labeled rhANXA5 with the FluoroTag™ FITC Conjugation Kit (Sigma-Aldrich®). Our home-made kit also contained PI solution (Sigma-Aldrich®) and a 10 × binding buffer (0.1 M HEPES-NaOH pH 7.4, 1.4 M NaCl, 25 mM CaCl₂). B16F10 melanoma cells (4X10⁴), a kind gift from Dr. Peter Henson (National Jewish Center for Immunology, Denver, CO, USA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), gentamicin 80 mg L⁻¹ (Novafarma, Anápolis, Brazil), and Fungisone 5 mg L⁻¹ (Bristol Myers Squibb, New York, NY, USA). To induce apoptosis, the cells were treated with cisplatin (Libbs, Embu, SP, Brazil) at different concentrations (0, 40, 80 and 160 μg/mL) and after 6 or 12 h, the cells were stained with our home-made kit or with a commercial kit from BD Biosciences (FITC Annexin Apoptosis Detection Kit II, BD Biosciences, San Diego, CA, USA). Cells were stained at a concentration of 10⁵ cells mL⁻¹ in 100 μL of 10X Binding Buffer using 5 μL of PI and 5 μL of rhANXA5 conjugated with FITC (home-made kit) or 5 μL of annexin-FITC from FITC Annexin Apoptosis Detection Kit II. All data were collected in a FACSCanto II flow cytometer (BD Bioscience) and analyzed using FlowJo software (Tree Stat, San Carlos, CA, USA).

AML cells were exposed to various doses of matrine with or without Baf A1 and rapamycin for 24 hrs in 24‐well plates at a density of 2 × 10⁵ cells/well. Then, apoptosis was determined using FITC Annexin Apoptosis Detection Kit II (BD Biosciences, San Diego, CA, USA), and cell cycle analysis was performed using Cell Cycle Staining Kit (MultiSciences, Hangzhou, China) on a flow cytometry (FACSCalibur; BD Biosciences) according to the manufacturer's protocols.

Flow cytometry and staining with propidium iodide and FITC Annexin V (BD Biosciences) was used to assess changes in viability and to track the mechanism of cell death after treatment. Cells were prepared and analyzed according to the FITC Annexin Apoptosis Detection Kit II (BD Biosciences). To analyze the influence of the compounds on glioblastoma and NHA distribution in cell cycle, cells fixed with 70% cold ethanol were stained with propidium iodide with addition of RNase (BD Biosciences) and analyzed. The extent of DNA DSBs measured by phosphorylation of H2A.X histone was obtained using Alexa Fluor 647 Mouse Anti-H2A.X (pS139) antibody (Becton Dickinson, San Jose, California, USA) after 48 h treatment with the compounds. Fixed cells were washed resuspended in 20 μl BD Perm/Wash™ buffer and stained for 20 min with H2A.X antibody (5 μl/test). All the experiments were performed using a FACS Canto II cytometer (Becton Dickinson, San Jose, California, USA).