Computational protocol: The circadian clock modulates anti-cancer properties of curcumin

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Protocol publication

[…] To image individual mitotic and apoptotic events a single field of view of cells maintained in FM and sealed in a 60-mm dish was captured at 5-min intervals using time-lapse videography continuously over 4–6 days. The imaging system consisted of an inverted microscope with a 20x objective lens, red LED light source, and a digital color camera. The imaging system was placed inside a 37 °C incubator to maintain ideal culture conditions. Time-lapse recording software FLIX (Nimisis.com) captured live cell events, and each image from the time series was analyzed by eye and with ImageJ software (National Institutes of Health). [...] Immunofluorescence staining was used to identify apoptotic cells and verify the peak and trough of the circadian rhythm in apoptotic events. C6 cells were fixed in 100 % methanol for 5 min and standard immunocytochemistry methods were used to identify cleaved caspase-3-positive cells. Anti-cleaved caspase-3 primary antibody (Cell Signaling) was used at 1:1000 dilution. The samples were rinsed after overnight incubation at 4 °C, and were then incubated for 2 h with Alexa488-conjugated, complementary secondary antibody. For nuclear staining, cells were stained with Hoechst 33342 (10–20 ng/ml, Invitrogen) for 2 min. Cells were imaged with a DMI3000B inverted fluorescence microscope (Leica Microsystems), a Rolera Thunder cooled-CCD camera with a back-thinned, back-illuminated, electron-multiplying sensor (Photometrics), X-Light spinning-disk confocal unit (CrestOptics), and a Spectra X LED light engine (Lumencore) with image acquisition and processing controlled by Metamorph software (Molecular Devices). Images were collected with a 40x objective lens along with standard DAPI and fluorescein filters. Images were further analyzed with ImageJ software. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Organisms Rattus norvegicus, Homo sapiens
Diseases Glioma, Neoplasms, Drug-Related Side Effects and Adverse Reactions
Chemicals Curcumin