Hoechst 33342

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Place aluminum foil over the sample to protect it from light and incubate at room temperature for 5-10 minutes. Optimize incubation time to minimize signal bleeding through to other channels.

Protocol tips
Place aluminum foil over the sample to protect it from light and incubate at room temperature for 5-10 minutes. Optimize incubation time to minimize signal bleeding through to other channels.

Publication protocol

Quantification of DNA compactness
Cells grown on a 96-well plate were washed with ice-cold PBS, fixed in 4 % formaldehyde for 20 min and washed twice with PBS. After the final PBS wash, the cells were stained with 1 μg/ml of Hoechst 33,342 solution in PBS for 10 min and subjected to analysis using an ArrayScan VTI HCS Reader (Thermo Fisher Scientific, Inc.) equipped with a 40× objective. To visually assess changes in chromatin structure after lactate or butyrate treatment, representative images were subjected to Sobel edge detection and thresholding using ImageJ software. The process of chromatin expansion decreases the number of distinct spaces within the nucleus, and these changes can be visualised via the detection of the Sobel edges as shown by Irianto and co-workers []. Quantitative analysis of chromatin compactness was performed by calculating the CV for the Hoechst intensity of all pixels within individual nuclei according to the method described by Contrepois and co-workers []. The Target Activation Bioapplication software (Thermo Fisher Scientific, Inc.) was set up to analyse 100 cells per well and to calculate the mean fluorescence and standard deviation of Hoechst staining within each nucleus. The CV parameter was obtained by dividing the standard deviation by the mean fluorescence. Each experiment was performed in six replicates.

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