For cell apoptosis detection, a FITC-Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA) was used according to the manufacturer’s instructions. Cells were rinsed with cold PBS and resuspended in 1 × binding buffer. Next, cells were dyed with 5 µl of Annexin V-FITC and 5 µl of propidium iodide in the dark at room temperature (RT) for 15 min. For cell cycle detection, cells were rinsed with cold PBS and fixed in 70% cold ethanol at 4 °C overnight. Afterwards, the rinsed cells were resuspended in a 500 µl mixture comprising 50 µg/ml propidium iodide and an equivalent volume of RNase A in the dark at RT for 30 min. A FACS Caliber (Beckman Coulter, USA) was used for acquiring the cells. FlowJo_V10 software was used for apoptosis analysis, and ModFit LT software was used for cycle analysis.
Facs caliber
Manufactured by Beckman Coulter
Sourced in United States
The FACS Caliber is a flow cytometry instrument designed for multi-parameter analysis of cells and particles. It utilizes four lasers and up to 16 fluorescent detectors to provide high-resolution data on a wide range of cellular properties and characteristics.
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7 protocols using facs caliber
1
Apoptosis and Cell Cycle Analysis
2
Measuring Mitochondrial Membrane Potential in A549 Cells
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A549 cells at density of 5 × 105 cells/mL were treated with 18.0 µM DEFL1 for indicated time to determine ΔΨm. Thereafter, the cells were collected, centrifuged at 600 g for 5 min and then washed with ice-cold PBS. Subsequently, cells were incubated with 40 nM DiOC6 (3) for 20 min in the dark at 37 °C. After that, stained cells were washed twice with ice-cold PBS once before being resuspened in 1 mL PBS. Finally, the quantity of DiOC6 (3) maintained 10,000 cells each sample was determined by a FACS Caliber flow cytometer (Beckman Coulter, Brea, CA, USA) at 484 nm of excitation wavelength and 501 nm of emission wavelength. The recorded data were analyzed by CellQuest software (BD Biosciences Inc., Franklin Lakes, NJ, USA) and expressed as a mean fluorescence intensity (MFI) [60 (link)].
3
Splenic and Lymph Node Cell Isolation
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Single-cell suspensions of splenic and inguinal nodes were prepared by perfusing mice with PBS, followed by staining with various antibodies and analyzing using flow cytometry on FACS caliber (Beckman). The eFluor 506 dye (eBioscience) was utilized to exclude dead cells. The stained cells were run on a flow cytometer (Beckman) using the CytExpert software v. 2.3 (Beckman) and then analyzed by FlowJo software (v. 10, TreeStar).
4
Cell Cycle Synchronization and Analysis
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Cells were starved 12 h for synchronization followed by re-stimulation with 10% FBS for 24 h. Cells were fixed with 75% ethanol and treated according to the Cell Cycle Detection Kit (BD Biosciences, Bedford, MA, USA). Cells were then sorted by a FACS Caliber flow cytometer (Beckman, CA, USA). The cell phase distribution was analyzed with Flowjo software (Treestar Inc., USA).
5
Cell Cycle Analysis of DT40 Cells
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Approximately, 0.5-1×106 of DT40 cells were harvested by centrifugation and re-suspended in 0.25 ml of PBS. 0.5 ml of −20°C absolute ethanol was then added drop-wise to the cells while gently vortexing. Cells were fixed for at least 30 min, the ethanol was removed, and the cells were washed twice with PBS and incubated in pre-warmed RPMI 1640 medium at 37°C for 30 min. After removal of the medium, the cell pellet was treated with RNase A at 100 μg/ml and propidium iodide at 40 μg/ml in PBS for 30 min. Flow cytometry was performed using FACS Caliber instrument (Beckman-Coulter).
6
Measuring Cellular ROS Production
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Briefly, DCF fluorescence intensity is directly related to the amount of ROS produced by the cells. After A549 cells were treated with DEFL1 for 24 h, 5 × 105 cells were harvested, washed with ice-cold PBS and incubated with DCFH-DA (50 μM of the final concentration) in the dark at 37 °C for 20 min. Subsequently, cells were washed twice with ice-cold PBS and resuspended in 1 mL PBS. ROS generation quantity was examined based on 10,000 cells each sample by a FACS Caliber flow cytometer (Beckman Coulter, Brea, CA, USA) at 488 nm of the excitation wavelength and 530 nm of the emission wavelength. The data were analyzed by CellQuest software (BD Biosciences Inc., Franklin Lakes, NJ, USA) and expressed as MFI [59 (link)].
7
Cell Cycle Analysis by Flow Cytometry
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RKO and HCT116 cells were analyzed for DNA content by FACS Calibur flow cytometer (Millipore, MA, USA) according to previously described methods [11 (link)]. Briefly, cells were synchronized, harvested, and fixed with 70% ethanol at 4 °C overnight. After washing with ice-cold PBS, cells were stained with propidium iodide (PI, 50 μg/ml), mixed with Triton X-100 and RNase A for 30 min, and cell cycle was analyzed by a FACS Caliber flow cytometer (Beckman Coulter, CA, USA).
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