After CdCl2 treatment, cells were washed with SF media and incubated with 5 M H2DCF-DA for 1 h at 37 C. Then cells were trypsinized, washed, and resuspended in 300 1 PBS. Intracellular ROS were measured by the DCF fluorescence in an Epics Elite flow cytometer (Beckman Coulter, Fullerton, CA). Alternatively (Fig. 6 B ), RMC (1 ×104 cells) were seeded into 96-well plates, grown overnight, and then starved in RPMI-1640 with 0.2 % FBS for 48 h. Cells were then washed and incubated with 100 M H2DCF-DA for 40 min in serumfree medium, washed again, and treated with the indicated concentrations of metals for 6 h. DCF was measured at excitation 485 nm and emission 535 nm with a SpectraMax i3 fluorescent plate reader (Molecular Devices, Sunnyvale, CA).
Epics elite flow cytometer
Manufactured by Beckman Coulter
Sourced in Canada
The Epics Elite flow cytometer is a high-performance instrument designed for advanced cellular analysis. It utilizes flow cytometry technology to rapidly measure and analyze the physical and fluorescent characteristics of individual cells or particles suspended in a fluid stream. The Epics Elite is capable of multi-parameter detection and can be used for a variety of applications, including but not limited to immunophenotyping, cell sorting, and research studies.
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Lab products found in correlation
Spectramax i3, by Molecular Devices (1 mentions)
Annexin 5 pe, by Keygen Biotech (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Annexin 5, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Human regulatory t cell staining kit, by Thermo Fisher Scientific (1 mentions)
Anticd34 qbend10, by Beckman Coulter (1 mentions)
12 protocols using epics elite flow cytometer
1
Measuring Intracellular ROS Levels
2
DNA Content Analysis by Flow Cytometry
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The cells that were used for DNA content analysis were trypsinized, pelleted by centrifugation (600 × g for 5 min), and resuspended in 500 μL of PBS. Ice-cold 70% ethanol (4.5 mL) was slowly added to each cell suspension while gently vortexing to inhibit clumping. Before flow analysis, the cells were repelleted, rinsed with PBS, and incubated for 30 min in a staining buffer containing 0.1% Triton X-100, 0.2 mg/mL RNase A, and 20 μg/mL propidium iodide. The cell cycle phase distribution was determined using a Beckman/Coulter EPICS Elite flow cytometer.
3
Apoptosis and Cell Cycle Analysis
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All pancreatic cancer cell lines were treated with ONC201 or ONC212 at the indicated doses and time-points. Post-treatment, both floating and adherent cells were collected, fixed in 70% ethanol and stained with propidium iodide in the presence of ribonuclease A. Flow cytometric data was collected using an EPICS Elite flow cytometer (Beckman-Coulter). The sub-G1 fraction (apoptotic) was quantified, and analysis was performed to quantify the distribution of cells in G1, S and G2-M phases of the cell cycle utilizing FlowJo software (FlowJo, LLC).
4
Cell Cycle Dynamics and Senescence in PSU-SK-1 Cells
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To study the growth dynamics of PSU-SK-1 in different passages, cell cycle studies were performed in cells from passages 5-30. Cells in the log phase of growth (second day of seeding) were trypsinized and fixed in cold 70% ethanol for at least 1 hour, then stored at -20°C. The thawed cells were washed with PBS, treated with RNAse (1 mg/ml) and stained with propidium iodide (50 mg/ml) for 30 minutes at 4°C. DNA content analysis was performed on an EPICS ELITE flow cytometer (Beckman Coulter). The cell cycle distributions were analysed using ModFit LT2.0 software.
β-galactosidase staining was performed to evaluate the amount of cells entering the senescence process. Cells grown in 6-well plates were washed with PBS (pH 7.2), fixed for 10 minutes with 4 % paraformaldehyde, and again washed with PBS (pH 7.2) plus 1 mM MgCl2. A volume of 1.2 ml of staining solution containing 1 mg/ml X-Gal, 0.12 mM K3Fe(CN)6, 0.12 mM K4Fe(CN)6, and 1 mM MgCl2 in PBS (pH 6.0) was added. After 6 hours of incubation at 37 °C, the staining was stopped by washing with PBS plus 1 mM MgCl2 (pH 7.2). The results were visualized under a light microscope.
β-galactosidase staining was performed to evaluate the amount of cells entering the senescence process. Cells grown in 6-well plates were washed with PBS (pH 7.2), fixed for 10 minutes with 4 % paraformaldehyde, and again washed with PBS (pH 7.2) plus 1 mM MgCl2. A volume of 1.2 ml of staining solution containing 1 mg/ml X-Gal, 0.12 mM K3Fe(CN)6, 0.12 mM K4Fe(CN)6, and 1 mM MgCl2 in PBS (pH 6.0) was added. After 6 hours of incubation at 37 °C, the staining was stopped by washing with PBS plus 1 mM MgCl2 (pH 7.2). The results were visualized under a light microscope.
5
Assessing Cell Cycle Profiles
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THP-1 cells (106/ml) were treated with media U. tomentosa and/or LPS for 24 h, exposed to ionizing radiation, and then incubated for 4–24 h, as indicated. The cells were fixed by suspension in 70% isopropanol at −20°C, and nuclear DNA was stained by incubation in 0.1% propidium iodide in methanol as described.[28 (link)] The fluorescent cell population profiles were created on a Beckman-Coulter Epics Elite flow cytometer (Beckman-Coulter, Mississauga, ON, Canada). The percent cells reported in each phase are the mean of three independent experiments.
6
Adenovirus-Induced Apoptosis and Cell Cycle
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Cells were infected with adenovirus for 72 h. Cell cycle and apoptosis were analyzed by flow cytometry. In brief, for apoptotic analysis, cells were stained with AnnexinV-PE and 7-ADD (KeyGenBiotech, China) following manufacturer's instructions. For cell cycle analysis, cells were fixed in 70% pre-cooling ethanol, and incubated with propidium iodide (PI) (Sigma, USA). Cell cycle and apoptosis were analyzed by a Beckman Coulter EPICS Elite flow cytometer. All tests were carried out in triplicates.
7
Bone Marrow Cell Cycle Analysis
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Single cell suspensions were prepared from the tibia and femur and analyzed for sub-G1 FACS analysis. Bone marrow cells were collected and fixed with 70% ethanol at 4°C. The samples were stained with propidium iodide (Sigma) in the presence of RNase and subjected to flow cytometric analysis using Epics Elite flow cytometer (Beckman Coulter, Fullerton, CA).
8
Cisplatin-Induced Cell Cycle Arrest
9
Apoptosis Assessment of MCF-7/HER2 Cells
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MCF-7/HER2cells were pretreated with cerulenin(2.5 mg/L), rapamycin (2.5 nmol/L) or the combination (2.5 mg/L cerulenin and 2.5 nmol/L rapamycin) for 12 h. The cells were washed twice with PBS and then were incubated in the dark for 15 min with binding buffer[10 mmol/L HEPES/NaOH (pH 7.4), 140 mmol/L NaCl and 2.5 mmol/L CaCl2], annexinV(200 g/L; BD Pharmingen) and propidium iodide (PI, 1 g/L; Sigma). The fluorescence of annexinV and PI was measured using FCM (flow cytometry, Epics Elite flow cytometer; Coulter). The data were analyzed using CellQuest.
10
Flow Cytometric Analysis of FoxP3+ Regulatory T Cells
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The cells were routinely fixed with 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) and stained with antibodies at 18°C. To test for intracellular FoxP3, lymphocytes were fixed and permeabilized using the Human Regulatory T Cell Staining Kit (eBioscience) following the manufacturer's instructions. The cells (at least 104 cells per test) were analyzed with an Epics Elite flow cytometer (Coulter, Marseille, France) in the logarithmic channel of fluorescence. The data were processed with EXPO32 software (Applied Cytometry Systems, Sheffield, UK). The gating strategy and controls for Figures 1 3 are described in Supplemental Figures 2 –4 .
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