Kaluza flow cytometry analysis software version 2

Peripheral blood mononuclear cells (PBMC) were isolated from 5ml fresh anti-coagulated blood by lymphoprep (StemCell Technologies) and blocked with FcR blocking buffer (Miltenyi Biotec, cat number: 130-059-901) at 4˚C for 10 min in the dark. Cells were then stained with fluorescein isothiocyanate (FITC) conjugated anti human CD19 (thermofisher, cat number: 11-0199-42) with either phycoerythrin (PE) conjugated anti human BR3 (thermofisher, cat number: 12-9117-42) or PE conjugated anti human TACI (thermofisher, cat number: 12-9217-42) at 4˚Cfor 30 min in the dark. Cells were acquired on a Navios flow cytometer (Beckman, U.S.). Data were analyzed by using Kaluza Flow Cytometry Analysis Software Version 2.0(Beckman, U.S.). For each test, at least 50,000 PBMCs were acquired. Mean fluorescent intensity (MFI) of eBR3 and TACIonCD19⁺ cells were determined. Data are represented as Mean ± SD.

One strain from each treatment was identified for further analysis of growth characteristics and pigment content. These strains were cultured in ASW under fluorescent light at 150 µmol photons m⁻² s⁻¹ with a 24:0 light cycle at 50 mL in 250 mL shaking tissue culture flasks (140 rpm) kept at 21 °C. Daily 1:2 dilutions were included to ensure optimal light and nutrient availability for 3 days, after which the flasks were placed in low light (< 10 µmol photons m⁻² s⁻¹) without dilution for an additional 5 days to maximise carotenoid content. Cell counts and chlorophyll fluorescence were measured daily using flow cytometry.
At the end of the experimental period, cultures were centrifuged, washed with MQ and flash-frozen in liquid nitrogen before being lyophilised and stored at − 80 °C for further analysis. Data visualisation was performed using GraphPad Prism version 9.0.2 for Windows (GraphPad Software, San Diego, California USA, http://www.graphpad.com) and Kaluza Flow Cytometry Analysis Software version 2.1 (Beckman Coulter, USA).
Biomass productivity was measured using the equation Productivity = Ln(N₂/N₁)/(t₂ – t₁) where N is found using absorbance at 750 nm. Fucoxanthin productivity was calculated by multiplying fucoxanthin content by N at any given time point.