The HT29 cells were seeded in 25 cm2 flasks at a density of 2 × 104 cells/cm2. The effects on the cell cycle were studied 48 h after treatment with 49 µM (R)-1 . To determine the cell cycle distribution at the end of incubation, HT29 cells were stained according to Busi et al. [23 (link)]. Briefly, 1 × 106 cells were pelleted and resuspended in trisodium citrate 0.1%, RNAse 0.1 mg/L, Igepal 0.01%, Propidium Iodide (PI) 50 µg/L. After 30 min at 37 °C in the dark, the cells were analyzed on a Coulter Epics Elite flow cytometer (Beckman Coulter) equipped with an argon ion laser tuned at 488 nm. PI fluorescence was collected on a linear scale at 600 nm and the DNA distribution was analyzed by the Modfit 5.0 software (ModFit Verity Software House http://www.vsh.com/ ).
Coulter epics elite
Manufactured by Beckman Coulter
The Coulter Epics Elite is a flow cytometry instrument designed for advanced research applications. It is capable of analyzing and sorting a wide range of cell types and particles. The Epics Elite provides high-speed analysis and precise data collection for multi-parameter detection and enumeration of cells.
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4 protocols using coulter epics elite
1
Cell Cycle Analysis of HT29 Cells
2
Determining Chironomid Genome Sizes
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The genome sizes of the two species were determined by flow cytometry (Cornette et al., in prep). Briefly, heads of adult chironomids were homogenized into a solution of 0.5% Triton X-100 in 1 ml phosphate buffered saline buffer, before staining the nuclei with 5 μg ml−1 of propidium iodide and filtering on a 30-μm mesh filter (Partec, Münster, Germany). The DNA content of stained nuclei was measured by a Coulter Epics Elite flow cytometry system (Beckman Coulter, Indianapolis, IN). The 2C DNA content of the sample was compared with the standard 0.36 pg DNA of D. melanogaster diploid nuclei.
3
Fluorescence-based Gametocyte Sorting
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Male and female gametocytes from the PfDynGFP, PfP47GFP and PfDynGFP/PfP47mCherry lines were sorted using the Coulter Epics Elite flow cytometer (Beckman Coulter) or the BD FACS Aria SORP flow cytometer keeping cells at 4°C in SA buffer. Gametocytes were first separated from uninfected red blood cells using forward and sideward scatters, followed by sorting males and females based on signal intensity of the fluorescent proteins (green fluorescent protein (GFP) or mCherry). An aliquot of sorted cells was reanalysed to determine purity of sorting.
4
DNA Content Analysis of Chironomid and Drosophila Nuclei
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Heads of Chironomid larvae and adults, and D. melanogaster adults (3 individuals per sample) were chopped with microscissors in a solution of 0.5 % Triton X-100 in 1 ml PBS buffer and digested for about 2 h at room temperature. The resulting lysate was then stained with 5 μg/ml of propidium iodide (PI) for several minutes and filtered with CellTrics Filter using mesh diameter of 30 μm (Partec) in order to collect nuclei. The DNA contents of dissociated nuclei were measured by a Coulter Epics ® Elite flow cytometry system (Beckman Coulter, Fullerton, CA) with the argon laser emitting 15 mW of exciting light at 488 nm. Fluorescent nuclei were detected using a high bandpass filter (610 nm). The C-value was calculated by multiplying the ratio of the mean fluorescence peak of the 2C sample/mean peak of the 2C standard with 0.18 pg, the haploid DNA content of D. melanogaster (Rasch et al., 1971 ). An example of the peaks obtained for P. vanderplanki and D. melanogaster nuclei is shown in Fig. 2.
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