Cultured cells were harvested 24 h after receiving 6 Gy of IR and then cell cycle progression and apoptosis were analyzed by flow cytometry. For cell cycle analysis, the cells were trypsinized and washed in PBS for 5 min prior to collection by centrifugation at 1500 rpm. The cells were then fixed with ice-cold 70% ethanol at −20°C overnight. The fixed cells were subsequently stained with 20 mg/mL propidium iodide (PI) staining buffer (containing 1% Triton X-100 and 100 mg/mL RNase A) for 30 min. DNA content was assessed using a FACSCalibur unit (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with ModFit LT v2.0 software. For the apoptosis assay, the cells were harvested by trypsinization and were washed with PBS. The cells were resuspended in 1× binding buffer at a concentration of 3 × 106 cells/mL. After staining the cells with fluorescein isothiocyanate (FITC) Annexin V and PI, the cells were analyzed using an Epics Profile II flow cytometer (Beckman Coulter, Fullerton, CA, USA) and Multicycle software (Phoenix Flow Systems, San Diego, CA, USA). All of the experiments were repeated at least three times.
Epics profile 2
Manufactured by Beckman Coulter
Sourced in United States
The Epics Profile II is a flow cytometer system designed for analyzing and sorting cells. It is capable of measuring multiple parameters, including size, granularity, and fluorescence, of individual cells within a sample. The Epics Profile II provides accurate and reliable data for a wide range of research and clinical applications.
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5 protocols using epics profile 2
1
Radiation-induced Cell Cycle and Apoptosis
2
Cell Cycle Analysis via Flow Cytometry
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Cells from each group were plated in a six-well plate at a concentration of 1×10 5 cells/ml, and they were collected after 48h incubation. Cells were washed with pre-cooling PBS for 2 times before they were fixed in 70% ethanol and stained with propidium iodide. The DNA content was analyzed with an Epics Profile II flow cytometer (Beckman Coulter) and analysis with Multicycle software (Phoenix Flow Systems). All experiments were repeated three times.
3
Endothelial Cell Attachment Analysis
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The HUVECs were cultured on the surface of the samples for 4 days (5 × 104 cells/mL) and 7 days (3 × 104 cells/mL). Thereafter, the cells were isolated and suspended by a flow cytometry staining buffer (00-4222-26, Thermo Fisher Scientific, USA). After blockage, FITC-CD31 labeled mouse antihuman monoclonal antibody was added. After rinsing twice, flow cytometry analysis was performed (FCM, EPICS Profile II, Coulter, USA).
4
HLA-A*0201 Peptide Antigen Presentation
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HLA-A*0201 Ag-processing defective T2 cell line was cultured in RPMI 1640 containing 10% FBS. Before use, the cells were incubated for 6 h at 37 °C in serum-free IMDM. Then, cells were washed once, suspended in serum-free IMDM containing 20 μM of 2-ME and 15 μg/ml of human β2-microglobulin (β2m) (Calbiochem, La Jolla, CA), and pulsed with 50 μM peptide. HLA-A2.1-restricted MAGE-2 CTL epitope KMVELVHFL (amino acid position in MAGE-2; 112–120) and Kb-restricted Hpa CTL epitope FSYGFFVI (amino acid position in Hpa; 519–526) served as positive and negative controls. After a 24 h incubation at 37 °C, T2 cells were washed once with cold PBS containing 0.5% BSA and 0.02% NaN3. They were then stained directly with primary anti-HLA-A2 Ab derived from BB7.2 and FITC-labeled goat-antimouse IgG (BD Biosciences Pharmingen, USA) secondary antibody. The percentage of FITC-positive cells as well as their staining intensity (mean fluorescence intensity (MFI)) was determined on an Epics Profile II (Coulter, Hialeah, FL). The Δ MFI for a particular mAb was calculated by subtracting the MFI of either the isotype-matched control mAb or the second-step Ab from each MFI value. The fluorescence ratio (FR) was calculated using the following formula: FR = (Δ MFI of peptide-treated T2 cells)/(Δ MFI of nontreated T2 cells).
5
Fas Expression Analysis in Granulosa Cells
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Harvested granulosa cells (2×107 cells) were resuspend in ice-cold wash buffer
[NaN3 (0.1%), FBS (0.5%) in PBS] and centrifuged (500 ×g; 5 min, 4℃). The cells
were immediately incubated in primary antibody solution [hamster anti-mouse Fas (0.5 µg/mL) in
wash buffer)] at 4℃ for 30 min., washed (500 ×g, 2×5 min in wash buffer), and incubated (30
min., 4℃ in dark), with FITC-conjugated mouse anti-hamster IgG (1.0 µg/mL). The cells were then
washed twice with the wash buffer, filtered through a 35 μm nylon mesh to remove cell clumps and
aliquoted for appropriate cell concentration (final concentration, 1×106 cells in 500
µL) for flow cytometric analysis (Coulter Epics Profile II, Hialeah, FL). Ten thousand cells
were counted in duplicates and analyzed by FACScan. As negative controls, nonspecific binding in
flowcytogram was determined in the absence of primary antibody.
[NaN3 (0.1%), FBS (0.5%) in PBS] and centrifuged (500 ×g; 5 min, 4℃). The cells
were immediately incubated in primary antibody solution [hamster anti-mouse Fas (0.5 µg/mL) in
wash buffer)] at 4℃ for 30 min., washed (500 ×g, 2×5 min in wash buffer), and incubated (30
min., 4℃ in dark), with FITC-conjugated mouse anti-hamster IgG (1.0 µg/mL). The cells were then
washed twice with the wash buffer, filtered through a 35 μm nylon mesh to remove cell clumps and
aliquoted for appropriate cell concentration (final concentration, 1×106 cells in 500
µL) for flow cytometric analysis (Coulter Epics Profile II, Hialeah, FL). Ten thousand cells
were counted in duplicates and analyzed by FACScan. As negative controls, nonspecific binding in
flowcytogram was determined in the absence of primary antibody.
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