Plan apochromat 100x 1.4na oil immersion objective

Flp-In-T-Rex HeLa cells were plated on coverslips pre-coated with poly-L-lysine for 24 hr. Asynchronously growing cells were fixed using 4% paraformaldehyde. Cells were stained for CREST/anti-centromere antibodies (1:100), diluted in 2% BSA-PBS for 1.5 hr. Goat anti–human Alexa Fluor 647 was used as secondary antibody. DNA was stained with 0.5 μg/ml DAPI and coverslips were mounted with Mowiol mounting media. Preparations were examined under a microscope (MARIANAS, from 3i-Intelligent Imaging Innovations, Inc.) built around a stand (Axio Observer Z1; Zeiss) equipped with CSU-X1 confocal scanner unit (Yokogawa Electric Corporation) and a Plan-Apochromat 100x/1.4NA oil-immersion objective (Zeiss). Images were acquired as z sections at 0.27 μm. Images were converted into maximal intensity projections, exported and converted into 8-bit. Quantification of kinetochore signals was performed on unmodified 16-bit z series images using Imaris 7.3.4 32-bit software. After background subtraction, all signals were normalized to CREST. At least 728 kinetochores were analyzed per condition. Measurements were exported in Excel (Microsoft) and graphed with GraphPad Prism 6.0.