Novel fluorochrome development enables 34-color high-content flow cytometry

Abstract

Abstract Flow cytometry is an essential technology to identify and quantify cell populations. Here we examined the development of new fluorochromes that could enhance flow cytometry’s capability. The latest spectral flow cytometers have 3 excitation lasers (405nm, 488nm, and 640nm) and incorporate the Avalanche Diodes Photodetector technology, which demonstrates significant improvement in sensitivity for the fluorescent emission signal longer than 800nm. However, there is no commercially available fluorochrome which could be excited by the above lasers and has peak emission signal beyond 800nm. To address the gap in technology, here we engineered 6 new fluorochromes: PE-750, PE-810, PE-830 for blue laser and APC-750, APC-810, APC-830 for red laser. These were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Then, the fluorochromes were conjugated with antibodies using Click chemistry, which is a simple, robust reaction with high-yield and high-reaction sensitivity. Also, we demonstrated long-term stability at −20°C with protein stabilizing cocktails. Most importantly, in order to show the utility of these novel fluorochromes, we created a 34-color flow cytometry panel to measure broad human immune function with high sensitivity on a 3-laser Aurora. The additional UV laser upgrade will increase the instrument capacity to more than 40 color. This panel will be applied to identify the expression patterns of many different receptors involved in trafficking on multiple different cell types from dissociated tumors and blood samples. In conclusion, this high-content flow cytometry panel will improve analysis and diagnosis in Immuno-oncology and facilitate innovation in biomarker discovery.