The cell cycle analysis were executed by following a previous report with slight modification [54 (link)]. In brief, A549 cells and H1299 cells (1 × 106 each) were seeded in 10 cm dishes and exposed to radiation or drug. The cells were then rinsed, trypsinized, and collected by centrifugation at 600 rpm for 5 min. To fix the cells, 70% precooled ethanol was added to cell pellets and kept at 4 °C overnight. The cells were then collected and treated with RNase A for 30 min at room temperature. After centrifugation, the cells were resuspended in 20 μg/mL of propidium iodide (PI) and sieved through a 37 m mesh. The DNA histogram was analyzed using a Beckman Coulter Cytomic FC500 flow cytometer and its bundled software (Beckman Coulter, Inc., Bream CA, USA). The sub-G1, G1, S, and G2/M phases were individually gated for quantification.
Cytomic fc500 flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Cytomic FC500 flow cytometer is a highly versatile instrument designed for a wide range of applications in clinical and research laboratories. It uses flow cytometry technology to accurately analyze and measure the physical and biochemical characteristics of cells, particles, and other biological samples. The Cytomic FC500 is capable of detecting and quantifying multiple parameters simultaneously, providing researchers and clinicians with valuable data and insights.
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8 protocols using cytomic fc500 flow cytometer
1
Cell Cycle Analysis of A549 and H1299 Cells
2
Cell Cycle Analysis of HeLa Cells
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For these experiments, HeLa cells were used, which were seeded in 6-well plates at a concentration of 5 × 105/well in a final volume of 2 mL culture medium. The cells were then treated with the test compounds for 24 h at the indicated concentrations. After this incubation period, the cells were detached with trypsin-EDTA and harvested by centrifugation. The pellet thus obtained was fixed in 70% ice-cold ethanol.
After this incubation period, the cells were detached with trypsin-EDTA and harvested by centrifugation. The pellet thus obtained was fixed in 70% ice-cold ethanol for at least 1 h. The cells thus fixed were treated with a 0.1% v/v solution of Triton_X-100 in phosphate buffered saline (PBS) containing RNAseA and propidium iodide (PI) at the final concentration of 0.02 mg/mL. The cells were incubated at room temperature for 30 min and then analyzed on a Cytomic FC500 flow cytometer (Beckman Coulter) in the FL3 channel. DNA histograms were analyzed using MultiCycle for Windows (Phoenix Flow Systems, San Diego, CA, USA).
After this incubation period, the cells were detached with trypsin-EDTA and harvested by centrifugation. The pellet thus obtained was fixed in 70% ice-cold ethanol for at least 1 h. The cells thus fixed were treated with a 0.1% v/v solution of Triton_X-100 in phosphate buffered saline (PBS) containing RNAseA and propidium iodide (PI) at the final concentration of 0.02 mg/mL. The cells were incubated at room temperature for 30 min and then analyzed on a Cytomic FC500 flow cytometer (Beckman Coulter) in the FL3 channel. DNA histograms were analyzed using MultiCycle for Windows (Phoenix Flow Systems, San Diego, CA, USA).
3
Cell Cycle Analysis via Flow Cytometry
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Cells were seeded, treated, and incubated for the indicated periods. The cell monolayer was harvested, washed in PBS, and fixed for 30 min in 1 mL of ice-cold 70% ethanol. The fixed cells were then pelleted, rinsed with PBS, incubated in the presence of RNase A for 15 min at 37 °C, stained with propidium iodide, and kept in the dark. The stained cells were analyzed for DNA content using a Cytomic FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Approximately 20,000 cells per sample were analyzed using excitation at 488 nm and emission at 617 nm. The percentage of cells in each phase of the cell cycle was determined using the CXP System Software (Beckman Coulter, Miami Lakes, FL, USA).
4
Cell Cycle Analysis of HeLa Cells
5
Cell Cycle Analysis of HUVECs
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HUVECs cells (5 × 105) were treated with different concentrations of the test compound for 24 h. After the incubation period, the cells were collected, centrifuged and fixed with ice-cold ethanol (70%). The cells were then treated with lysis buffer containing RNAse A and 0.1% Triton X-100, and then stained with propidium iodide (PI). Samples were analyzed on a Cytomic FC500 flow cytometer (Beckman Coulter). DNA histograms were analyzed using MultiCycle for Windows (Phoenix Flow Systems).
6
Cell Cycle Analysis by Flow Cytometry
7
Cell Cycle Response to Radiation
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Cells were plated at 40–50% confluency in 75 cm2 tissue culture flasks and allowed to adhere overnight followed by radiation exposure from 0 to 8 Gy. After irradiation at the indicated doses, cells were grown for 0–96 h and harvested. Samples were trypsinized, resuspended in PBS, and fixed with cold 75% ethanol overnight at 4 °C. The fixed samples were centrifuged at 2,200 rpm for 5 min, then washed twice with cold PBS and resuspended in 500 μL of PI staining solution (BD Biosciences) for 30 min. The DNA content of labeled cells was measured by Cytomic FC 500 flow cytometer (Beckman Coulter, Inc).
8
Immunophenotyping of Human Mesenchymal Stem Cells
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Immunophenotyping of hMSCs was performed using human PE-conjugated monoclonal antibodies (Miltenyi Biotech, UK) specific for CD14 (clone: Tük4/ catalog No.130-113-709), CD19 (clone: LT19/ catalog No. 130-113-731), CD34 (clone: AC136/catalog No. 130-113-741), CD45 (clone: 5B1/catalog No. 130-113-680), CD73 (clone: AD2/catalog No. 130-097-943), CD90 (clone: DG3/catalog No. 130-117-537), CD105 (clone: 43A4E1/catalog No. 130-098-906), HLA-DR (clone: AC122/catalog No. 130-098-177). Mouse IgG1 (clone: IS5-21F5/catalog No. 130-113-762) and IgG2a (clone: S43.10/catalog No. 130-113-834) were used for isotype controls. Briefly, 1 × 105 hMSCs were aliquoted into individual microcentrifuge tubes, washed with incubation buffer (0.075% EDTA/0.5% BSA in PBS) and centrifuged for 5 min at 300g. Cell pellets were re-suspended in 100 µl of specific antibody solution followed by incubation at 4°C for 10 min. Labeled hMSCs were washed in a 10× volume of incubation buffer and centrifuged at 300g for 10 min. The supernatant was aspirated and cell pellets re-suspended in 200 µl incubation buffer for analysis on a Cytomic FC500 flow cytometer (Beckman Coulter, UK) and Cyflogic v.1.2.1(CyFlo Ltd, UK).
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