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Description

Sub-confluent, interphase Human Umbilical Vein Endothelial Cells (HUVECs) transfected with GFP-WT-MCAK (Mitotic Centromere Associated Kinesin) and grown in culture for 8 hours. Cells were fixed in PHEM buffer containing 4% paraformaldehyde, 0.05% gluteraldehyde, and 0.2% Triton X-100. Cells were then labeled with mouse anti-tubulin detected by goat anti-mouse Alexa 568 antibody. Cells were imaged on a spinning disk confocal microscope using a 60x 1.4 NA oil immersion objective lens on a Nikon Eclipse Ti equipped with Perfect Focus System and a Yokogawa CSU-X spinning disk confocal scan head equipped with a multi-bandpass dichromatic mirror (Semrock; Rochester, NY) and bandpass filters (Chroma; Rockingham, VT) in an electronic filterwheel for selection of GFP or Texas red emission. Excitation light provided by a custom-built laser combiner module (modification of LMM-3, Spectral Applied Research; Richmond Hill, Ontario, Canada) containing 500mW solid state lasers (488 nm: Coherent; 561 nm: MPB Communications; Montreal, Quebec) that were shuttered with electronic shutters and attenuated and/or directed to a fiber-coupled output port with an AOTF (Neos Technologies, Melbourne, FL) and directed to the confocal scan-head via a single-mode optical fiber (Oz Optics, Ottawa, Ontario, Canada). A Coolsnap HQ2 camera captured images with 200ms exposure @ 488nm and 400ms exposure @ 560nm.

Biological Sources

NCBI Organism Classification
Homo sapiens
(human)
Cell Type
endothelial cell
primary cell line cell
Cellular Component
kinesin complex
microtubule

Biological Context

Biological Process
protein localization to chromosome, centromeric region

Attribution

Names
Kenneth Myers
Clare M. Waterman
Other
Laboratory of Cell and Tissue Morphodynamics Cell Biology and Physiology Center National Heart, Lung, and Blood Institute National Institutes of Health Building 50 South Drive Room

Dimensions

Spatial Axis Image Size Pixel Size
X 1392px 105μm
Y 1040px 105μm
Channel Wavelength
1 488 nm
2 561 nm
*CIL – Cell Image Library accession number. Please use this to reference an image.