Five-laser Aurora spectral flow cytometer

PROJECT SUMMARY Flow cytometry is an essential tool for the single cell analysis of a wide variety of cell types to assess the expression of surface and intracellular markers and perform functional measures. Conventional flow cytometry has been unable to leverage the large variety of fluorochromes available for marker analysis, because the technology is focused on a narrow window of their emission spectrum, in which many of these fluorochromes overlap, limiting the number of markers that can be measured. In contrast, spectral flow cytometry acquires the full emission spectrum of each fluorochromes, which enables high resolution characterization of fluorochromes (or fluorescent proteins) that can be used in conjunction. Moreover, the autofluorescence emitted by unstained cells can also be acquired and subtracted for better detection of weaker fluorescent signals. The 5-laser Aurora from Cytek is one of the most powerful spectral flow cytometry instruments currently available and the need for this technology is supported (1) by the fact that our two most high-end conventional flow cytometers (LSRII) are reaching end of serviceable life in one year, and (2) by increasing demand for greater capabilities, including more parameters analyzed per sample, as well as better sensitivity for dim markers and for small particles. With the 5-laser Aurora, investigators can expand the number of markers assessed to well beyond 30 markers: Cytek has validated a 40-color panel for human blood and Dr. Farber at the Columbia University Irving Medical Center (CUIMC) is applying panels of 25-35 parameters for several of her projects with several substitutions made possible by the flexibility of panel design. This capability is attractive to many Flow Cytometry Core users who have limited amount of precious material to work with, including cells isolated from small tissue biopsies, humanized mice (particularly blood and lymph node samples), patient airway washes and tissue organoids. This technology is also ideal as an upgrade option for most of our LSRII users, as opposed to mass cytometry, which requires acquiring or generating all new reagents. When testing the Aurora, users have been able to overcome major roadblocks in their projects, such as the ability to identify tissues cells whose autofluorescence was masking the expression of a fluorescent reporter. Users also validated the detection of particles or cell products in the 100-200 nm size range, made possible by the Aurora's violet side scatter detector. In this proposal, 34 principal investigators at CUIMC have indicated how their projects (84% of which are NIH- funded) will be significantly enhanced by upgrading from conventional flow cytometry instrument to the 5-laser Aurora. Thus, the addition of this instrument will have a beneficial and long-lasting impact on biomedical research encompassing a broad variety of fields. Generous institutional support is provided in the form of incentives and coverage of maintenance costs, and the operation of the 5-laser Aurora will be sustainable in the long-term because its maintenance is comparable to conventional instruments in use.