Comparison of mean separation distances measured using Delta analysis in live and fixed metaphase HeLa cells. (A and B) HeLa cells transfected with cDNA plasmid pairs encoding Hec1-GFP and mCherry–CENP-C (A) or Zwint1-GFP and Hec1-tdTomato (B) were subjected to two-color live imaging (A and B, top) or indirect immunofluorescence using the anti-GFP antibody (A and B, bottom) and either the anti-mCherry antibody (A, bottom) or the anti-RFP antibody (B, bottom). Bars, 5 µm. (right) Images are boxed regions within the cells in the top and bottom of A and B magnified to show representative sister kinetochore pairs used for Delta analysis. Bars, 1 µm. (C) Mean Delta values measured from live and fixed cells in A and B.