For the leukemic HL60 cell line, the cells were treated with the mono and combined IC 50 doses of Vorinostat and PLX51107 for 72 hours; whereas non-leukemic control cells were treated for 48 hours and "BD Cycletest Plus DNA Reagent Kit (Becton Dickinson, Cat# 340242)" was used for cell cycle arrest analysis. The effects of the treatments on the cell cycle were evaluated in the ow cytometry (BD Accuri™ C6 Plus ow cytometer) device.
Accuri c6 plus ow cytometer
Manufactured by BD
Sourced in United States
The BD Accuri C6 Plus flow cytometer is a compact and user-friendly instrument designed for routine analysis of cells and particles. It features a solid-state laser and advanced optics to provide high-quality data in a small footprint. The core function of the BD Accuri C6 Plus is to enable the detection, identification, and quantification of various cell populations based on their size, granularity, and fluorescent properties.
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5 protocols using accuri c6 plus ow cytometer
1
Cell Cycle Analysis of Leukemic HL60 Cells
2
Adv-lnc-GULP1-2 Regulates Granulosa Cell Cycle
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Primary granulosa cells treated with 5×10 10 PFU/ml adv-control or adv-lnc-GULP1-2:1 (Hanbio Biotechnology, Shanghai, China) for 48h were seeded (70% con uent) in 12-well plates for ow cytometry analysis. Cells were digested with EDTA-free trypsin, washed twice with cold PBS (1000 rpm × 5 min), and then xed with 70% ethanol at 4 ℃ overnight. The xed cells were centrifuged (1000 rpm × 5 min) again on the next day, resuspended in 500μl of PI/RNase solution KeyGen Biotech Nanjing China), and then incubated in the dark for 30 minutes at 37 ℃. Flow cytometry studies were performed by using BD Accuri C6 Plus ow cytometer (BD Biosciences, San Jose, CA). The percentage of cells in different phases of cell cycle were analyzed by using FlowJo 10.4 (BD Biosciences).
3
Adenoviral Regulation of Granulosa Cell Cycle
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Primary granulosa cells treated with 5×10 10 PFU/ml adv-control or adv-lnc-GULP1-2:1 (Hanbio Biotechnology, Shanghai, China) for 48h were seeded (70% con uent) in 12-well plates for ow cytometry analysis. Cells were digested with EDTA-free trypsin, washed twice with cold PBS (1000 rpm × 5 min) and then xed with 70% ethanol at 4 ℃ overnight. The xed cells were centrifuged (1000 rpm × 5 min) again on the next day, resuspended in 500μl of PI/RNase solution KeyGen Biotech Nanjing China), and then incubated in the dark for 30 minutes at 37 ℃. Flow cytometry studies were performed by using BD Accuri C6 Plus ow cytometer (BD Biosciences, San Jose, CA). The percentage of cells in different phases of cell cycle were analyzed by using FlowJo 10.4 (BD Biosciences).
4
Apoptosis Detection by Flow Cytometry
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The mode of cell death was determined using ow cytometry. Apoptotic cell death was detected using an Annexin V-Flourescein isothiocyanate (FITC)/ Propidium iodide (PI)-PE (phycoerythin) apoptosis detection kit (abcam) as described previously. Brie y, cells were collected, incubated with 1% Annexin V and 1% PI in binding buffer, and then analyzed directly using uorescence-activated cell sorting (FACS, Accuri C6 plus ow cytometer, BD Bioscience, Franklin Lakes, NJ, USA).
5
Sperm Oxidative Stress and Phosphatidylserine
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ROS levels were assessed according to the experimental protocol of Guthrie and Welch (Guthrie &Welch 2010). Brie y, ROS in the sperm was detected using H2DCFDA. The level of ROS was evaluated by measuring the uorescence intensity of H2DCFDA in the live sperm population. All samples were analyzed to derive their uorescence signals using a BD Accuri ™ C6 Plus ow cytometer. The data are expressed as the percentage of ROS molecules measured in live sperm. The PS translocation status was assessed through the annexin V-FITC detection Kit. Brie y, spermatozoa were pelleted twice using PBS at 300 × g for 5 min, and then diluted in 1 mL of 1 × annexin buffer (5 × 10 6 sperm/mL). From this suspension, 100 µL was collected in new 1.5 mL tubes, and mixed with 5 µL annexin-FITC stain and 5 µL propidium iodide (Malo et al.) . The mixture was maintained in the dark at room temperature (25°C) for 15 min. Thereafter, 400 µL of 1 × annexin buffer was mixed to the tubes and analyzed by ow cytometry.
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