Quantitative Analysis of the Cell Cycle in Imaging Applications ( h A0781) a DNA topoisomerase alpha in...)

Quantitative Analysis of the Cell Cycle in Imaging Applications

h, A0781), a DNA topoisomerase alpha inhibitor (G1/S blocker) for two hours or colcemid (Sigma-Aldrich Cat # D7385) a (G2/ M blocker for two hours. During drug treatment, cells were cocultured with 20 μM BrdU (Calbiochem, 203806) (a thymidine analogue that incorporates into DNA) for the final hour of culture.

For image analysis, cells were fixed and permeabilized in BD CytoFix/CytoPerm (4% paraformaldehyde with 0.1% saponin, Cat. No. 552598a) for 20min, washed with BD Perm/Wash Buffer (PBS with 0.1% saponin and 3% fetal calf serum, FCS, Cat. No. 552598b), and permeabilized again in BD CytoPerm Plus Permeabilization Buffer (BD 552598c) for 10 min. Afterwards, they were washed with the Perm/Wash Buffer, and re-fixed for 5 minutes. Cells were then washed with Perm/Wash Buffer, treated with DNAse (Sigma-Aldrich, D4513, 15μg/well in PBS at 37C for 1 hour), washed in Perm/Wash Buffer and stained simultaneously with Alexa Fluor 488 (Invitrogen, A-20000) conjugated anti-BrdU (Cat. No. 555627). Alexa Fluor 647 conjugated anti phospho-Histone H3 (Serine 28) (Cat. No. 558217), and Alexa Fluor 555 (Invitrogen A- 20009) conjugated anti-Ki-67 (Cat. No. 610969) in PBS with 3% FCS at room temperature for 1 hour. Cells were washed three times in Perm/Wash Buffer and stained with 2 μg/mL Hoechst 33342 (Invitrogen, H3570), in Perm/Wash Buffer

Images were acquired on a BD Pathway Bioimager and analyzed using Attovision software and BD Image Data Explorer.

Cells were imaged using a 20 (0.75 NA) objective and analyzed using the built-in tools of the software package. Numerical data was imported into the Microsoft Excelbased BD Image Data Explorer where the cell population was analyzed for nuclear fluorescence intensity by setting a threshold for the three fluorescence channels. The threshold was set on the nuclear intensity of the BrdU and phospho-Histone 3 negative cells.

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