Cytoflex lx cytexpert software

In all cases, manufacturer quality control programs were run and passed before acquisition of data. Between 5000 and 10,000 gated lymphocytes were acquired at low differential pressure (10–12 μl min⁻¹) on all available detectors on a CytoFLEX LX/ CytExpert software (6 laser: UV3‐V5‐B3‐YG5‐R3‐IR2, Beckman Coulter) and an Aurora 5L/SpectroFlo® software (5 laser: UV16‐V16‐B14‐YG10‐R8, Cytek, Fremont, CA, USA).
Raw FCS data files were used to calculate SI from both CytoFLEX and Aurora cytometers. Additionally, for Aurora, each fluorochrome was unmixed independently with and without AF extraction using unstained lymphocytes as a reference for AF via SpectroFlo. Henceforth, unmixed, AF extracted data is referred to as (U‐AF) and unmixed, no AF extraction is referred to as (U). CD4⁺ SI were calculated based on the mean fluorescence intensity (MFI) for both CD4⁺ and CD4⁻ lymphocytes and the standard deviation (SD) of CD4⁻, based on Equation (10):
Normalized stain index (SInorm) at each detector was additionally calculated based on calculated SI per detector and maximum stain index value across all detectors for each fluorochrome (SI_max), based on this equation: $${\mathrm{SI}}_{\mathrm{norm}}=\frac{\mathrm{SI}}{{\mathrm{SI}}_{\max }}.$$