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DAPI staining and fluorescence quantification

HS Handuo Shi
YH Yan Hu
PO Pascal D. Odermatt
CG Carlos G. Gonzalez
LZ Lichao Zhang
JE Joshua E. Elias
FC Fred Chang
KH Kerwyn Casey Huang
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Cells were grown in the same conditions as in single-cell imaging experiments. At each time point, 1 mL of each sample was taken, pelleted at 6500g for 1 min, and fixed via resuspension in 500 µL 70% ethanol and incubation for 15 min at room temperature. Cells were then pelleted at 6500g for 1 min, resuspended in 500 µL PBS with 4′,6-diamidino-2-phenylindole (DAPI) added to a final concentration of 1 µg/mL, and incubated in the dark for 15 min. Cells were washed with PBS twice by pelleting at 6500g for 1 min followed by resuspension. Cells were spotted onto 1% agarose pads and imaged in phase contrast and fluorescence using a DAPI filter. DAPI fluorescence was quantified by summing the intensity values of each pixel within the cell contour, and then normalizing to the corresponding intensity in the control.

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