Flow cytometry has been around since the 1950s when Wallace Coulter developed the first flow cytometry device and fluorescence-based flow cytometry was introduced in 1968 by Wolfgang Göhde. Since then, fluorescence-based flow cytometry and fluorescence-activated cell sorting (FACS) have blown up to become a mainstay of analytical scientific approaches in every field of cell biology, especially immunology. However, the dominance of fluorescence-based flow cytometry for analytical cellular biology may change with the recent introduction of a new technology: Time of Flight Mass Cytometry (CyTOF).
In fluorescence-based flow cytometry, cells or particles labeled with fluorescent dye-conjugated antibodies or other fluorescent proteins flow in a single file stream past a series of lasers that emit light at specific wavelengths, causing the fluorescent dyes to become excited and emit light caught by detectors. Thus, a quantitative measure of intensity for each fluorescent parameter, pertaining to the expression level of the antibody-targeted antigen of interest, is obtained for every cell. The BD Biosciences Influx cell sorter is currently a top of the line fluorescence-based flow cytometer, and supports up to 10 lasers and detection of up to 24 parameters. However, even with a thorough understanding of flow cytometry, the actual number of utilizable parameters will be typically be far less due to limitations including spectral overlap of fluorescent dyes.
CyTOF utilizes an entirely different technique to quantify protein expression levels on a single cell level: the use of transition element isotopes to label antibodies. The quantities of isotopes bound to each cell are then analyzed by a time-of-flight mass spectrometer. While compensation issues due to spectral overlap between fluorophores limits the effective number of parameters assessable by fluorescence-based flow cytometry to far below the theoretical maximums, CyTOF does not suffer from these limitations as there is no requirement for compensation. In addition, as the metal isotopes used are rare, there is no autofluorescence of cells, another limitation of fluorescence-based flow cytometry. Proof of principle studies have been published by Gary Nolan and colleagues at Stanford University, and have demonstrated the simultaneous use of 34 cell surface and intracellular parameters. The CyTOF instrument can theoretically detect up to 100 isotopes, thus far extending the ability of researchers to simultaneously assess the expression of many more proteins per cell.
The CyTOF instrument is commercially available from DVS Sciences. DVS Sciences also offers an expanding list of pre-conjugated metal isotope-labeled antibody reagents and additionally a MAXPAR® labeling kit for conjugation of other antibodies to 33 different metals, allowing researchers to select many additional antigens of interest for analysis.
I have previously stressed the importance of studying cell biology on the single cell level in order to understand the relationships that occur between expression of proteins and signaling states in unique cell populations and on the single cell level. The addition of CyTOF to the reseracher’s arsenal will allow these types of questions to be addressed on an even more complex level.
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Tricks for analyzing PBMC populations by flow cytometry
photo credit: PNNL – Pacific Northwest National Laboratory via photopincc