With the arrival of full spectrum flow cytometry and the possibility to detect up to 40 markers at the single-cell level, there has been a paradigm shift when designing, optimising and analysing high-dimensional full spectrum flow cytometry data. However, not enough is known regarding the implications of how an increasing number of lasers affects the resolution and detection of high-dimensional flow cytometry panels.
A 24-colour panel on mouse splenocytes was run on a 3L (Violet (V): 405nm, Blue (B): 488nm and Red (R): 640nm), 4L (V, B, R and Yellow-Green (Y/G): 561nm and 5L (V, B, R, Y/G and Ultraviolet (UV): 355nm) full spectrum flow cytometer (Cytek Aurora). We observed that the saturation point of all antibodies was the same in all systems used, but that the stain index differed depending on the laser configuration tested and produced variable spread in some markers included in the panel. Additionally, we demonstrated that although autofluorescence (AF) increases in complexity as more lasers were added to the system, this provided additional information to distinguish the autofluorescence signature of different cell types and assist with improved autofluorescence extraction.
In summary, we could determine that the laser configuration plays an important role in full spectrum panel design and directly affects the fluorochrome stain index, fluorochrome spread and autofluorescence detection/extraction. These parameters and decisions on panels design are therefore important additional factors to consider when deciding the appropriate laser configuration for new instrument purchases.