Comparison of three cell fixation methods for high content analysis assays utilizing quantum dots
Article first published online: 25 SEP 2008
© 2008 The Authors Journal compilation © 2008 The Royal Microscopical Society
Journal of Microscopy
Volume 232, Issue 1, pages 91–98, October 2008
How to Cite
WILLIAMS, Y., BYRNE, S., BASHIR, M., DAVIES, A., WHELAN, Á., GUN’KO, Y., KELLEHER, D. and VOLKOV, Y. (2008), Comparison of three cell fixation methods for high content analysis assays utilizing quantum dots. Journal of Microscopy, 232: 91–98. doi: 10.1111/j.1365-2818.2008.02083.x
- Issue published online: 25 SEP 2008
- Article first published online: 25 SEP 2008
- Received 8 January 2008; accepted 5 March 2008
- Cell lines;
- high content screening;
- quantum dots
Semiconductor nanoparticles or quantum dots are being increasingly utilized as fluorescent probes in cell biology both in live and fixed cell assays. Quantum dots possess an immense potential for use in multiplexing assays that can be run on high content screening analysers. Depending on the nature of the biological target under investigation, experiments are frequently required on cells retaining an intact cell membrane or also on those that have been fixed and permeabilized to expose intracellular antigens. Fixation of cell lines before or after the addition of quantum dots may affect their localization, emission properties and stability. Using a high content analysis platform we perform a quantitative comparative analysis of three common fixation techniques in two different cell lines exposed to carboxylic acid stabilized CdTe quantum dots.
Our study demonstrates that in prefixed and permeabilized cells, quantum dots are readily internalized regardless of cell type, and their intracellular location is primarily determined by the properties of the quantum dots themselves. However, if the fixation procedures are preformed on live cells previously incubated with quantum dots, other important factors have to be considered. The choice of the fixative significantly influences the fluorescent characteristics of the quantum dots. Fixatives, regardless of their chemical nature, negatively affected quantum dots fluorescence intensity. Comparative analysis of gluteraldehyde, methanol and paraformaldehyde demonstrated that 2% paraformaldehyde was the fixative of choice. The presence of protein in the media did not significantly alter the quantum dot fluorescence.
This study indicates that multiplexing assays utilizing quantum dots, despite being a cutting edge tool for high content cell imaging, still require careful consideration of the basic steps in biological sample processing.