Expo32 adc software

The apoptosis of murine primary chondrocytes was measured by Annexin-V-FITC/PI staining assay (Thermo Fisher Scientific, Inc.). Primary chondrocytes cultured in 12-well plates (5×10³ cells/well) subjected to different treatments were stained with 5 µl Annexin V-FITC and 5 µl PI. The mixture was incubated for 15 min at room temperature in the dark. The percentage of apoptotic cells (Annexin V-FITC⁺/PI⁻) was calculated using a flow cytometer (BD Biosciences) according to the manufacturer's instructions. Data were acquired via flow cytometry (Beckman Coulter, Inc.). Fluorescence analyses were performed on COULTER EPICS XL Flow Cytometer (Beckman Coulter, Inc.) using Expo32-ADC software (version 1.2B; Beckman Coulter, Inc.).

Flow cytometric analysis was performed to confirm the types of cells using specific
surface antigens of MSCs (CD29 and CD90) and HSCs (CD34 and CD45). The cells in
culture were trypsinized and stained with fluorescein isothiocyanate (FITC) or
phycoerythrin (PE) conjugated antibodies, including FITC-CD34, PE-CD90, FITC-CD45,
and PE-CD29 monoclonal antibodies (Immunotech, USA). Ten thousand labeled cells were
acquired and analyzed using a Becton Dickinson flow cytometer (USA). Data (collected
in list mode) were analyzed using the EXPO32 ADC software (Beckman Coulter,
Germany).

MDA-MB-231 cells were cultured in 2% FBS in the presence or absence of 150 ng/ml rHuPCSK6 for 24 h and stained using an Annexin V-fluorescein isothiocyanate apoptosis detection kit (BD Biosciences), according to the manufacturer's protocol. Cells were detected using a Coulter Epics XL flow cytometer, and DNA content was analyzed with Beckman Coulter EXPO32 ADC software (version 1.2B; both Beckman Coulter, Inc.). The experiments were repeated 3 times in triplicate.

Intracellular peroxide levels were detected by flow cytometric analysis using an oxidation-sensitive fluorescein labeled dye, carboxylated dichlorodi-hydrofluorescein diacetate (carboxy-H2DCFDA, Molecular Probes, Carlsbad, CA, USA) (15 (link)). Upon oxidation by intracellular ROS, the non-fluorescent dye is converted into its fluorescent form. INS-1 cells were labeled with 100 M carboxy-H2DCFDA for 1 hour at 37°C. Following cell loading of the dye, the cells were washed twice with PBS and then put back into culture conditions for 2 hours. INS-1 cells were then harvested, washed twice with PBS, and re-suspended in trypsin–EDTA (0.25% trypsin, 2 mM Na4-EDTA, Invitrogen) for 5 minutes at 37°C. In order to disperse the cells into a single cell suspension, INS-1 cells were gently passed 20 times in and out of a 200–1,000 lL tip. The cells were then washed twice with ice-cold PBS. Analysis of cells was performed using a 488 nm argon laser EPICS XL-MCL flow cytometer controlled by EXPO 32-ADC software (Beckman Coulter, Fullerton, CA, USA). ROS values were analyzed based on fluorescence intensity.