Computational protocol: Assembly of Drosophila Centromeric Chromatin Proteins during Mitosis

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Protocol publication

[…] In duplicate experiments, exponentially growing clonal S2 cells stably expressing SNAP-tagged centromeric proteins and GFP-tubulin were incubated in 300 µl of serum containing medium (SM) containing 4 µM tetramethylrhodamine (TMR) for 15 min. After 3 washes with 5 ml of SM, cells were counted, diluted to 1×106 cells/ml and plated in a 12 well plate. Samples were taken, counted, fixed and mounted right after TMR (Day 0) and after 24 h (Day 1). Cell counting confirmed that cell numbers doubled during the 24 h period. Cells were imaged immediately after mounting and 10 fields of cells (200–300 cells) were acquired on a PersonalDV microscope (Applied Precision) using a 60×/1.42 Olympus oil immersion objective. Increments in z were set at 0.3 µm, sample thickness was 11 µm, and the bin was set to 2×2. 100× bin 1×1 images were also acquired to make the figures. All images were scaled in Softworks, maintaining the parameters constant between samples, saved as .psd files and figures were assembled in Adobe Illustrator. [...] 5×105 cells were centrifuged for 5 min at 600 g at room temperature and resuspended in 150 µl of PBS. 350 µl of ice-cold 100% 200 proof ethanol was added drop wise while vortexing cells gently. Cells were incubated at 4°C for 24 h, washed twice with 1 ml of PBS and then resuspended in 1 ml of PBS-T containing 20 µg/ml Propidium Iodide and 0.2 mg/ml RNAse A and incubated at 37°C for 15 min. Samples were analyzed on a Beckman-Coulter EPICS XL flow cytometer and the data was analyzed in FlowJo. Approximate percentage of cell in each cell cycle phase was estimated using the Watson-pragmatic model in Flowjo, eliminating doublets resulting from cells in cytokinesis/G1. […]

Pipeline specifications

Software tools Adobe Illustrator, FlowJo
Applications Miscellaneous, Flow cytometry
Organisms Drosophila melanogaster, Homo sapiens