Cell quest data acquisition software

The binding of plant-made rituximab to target cells (Wil2-S) was determined with flow cytometry analysis using a Becton Dickinson Accuri C6 flow cytometer. Wil2-S lymphoma cells at 1×10⁶ cells/mL were incubated with different concentrations of Rituxan^® (Genentech, South San Francisco, CA, USA), or plant-made rituximab treated either with 5 µM, 0.25 µM or no kifunensine for 45 min at 4 °C. Cells were washed and incubated with FITC conjugated anti-human IgG Fc (BioLegend, San Diego, CA, USA) in phosphate-buffered saline (PBS) with 2% fetal bovine serum (FBS). FITC-labeled Mouse IgG2a, kappa (BioLegend, San Diego, CA, USA) was used as an isotype control. Cells were analyzed by flow cytometry after briefly washing them with PBS containing 2% FBS. 7-AAD (7-amino-actinomycin D) exclusion dye was used for the quantification and segregation of dead cells in each sample. Cell Quest data acquisition software (BD Biosciences, San Jose, CA, USA) and Flowjo FACS (Fluorescence-activated cell sorting) analysis software (Tree Star Inc., Ashland, OR, USA) were used to derive data plots. Cell labelling and Flowjo FACS analysis was as described earlier [45 (link)].

Flow cytometry analysis was performed to determine and compare the binding of _NbRTX^A2G2 and Rituxan^® (Genentech, South San Francisco, CA, USA) antibodies to target cells (Wil2-S and Daudi) using a FACS Calibur (BD Biosciences, San Jose, CA, USA). Briefly, 100 µL cells at 1 × 10⁶ cells/mL were incubated with different concentrations of Rituxan^® standard and _NbRTX^A2G2 for 30 min at 4 °C. Cells were then washed and incubated with 5 µL of FITC anti-human IgG Fc (BioLegend, San Diego, CA, USA). FITC Mouse IgG2a, k Isotype Ctrl (FC) (BioLegend, San Diego, CA, USA) was used as isotype control. Cells were washed with stain buffer and analyzed by flow cytometry. Nucleic acid dye 7-AAD was used for the quantification of dead cells in each sample as 25 ng/500 µL. Cell Quest data acquisition software (BD Biosciences, San Jose, CA, USA) and Flowjo FACS analysis software (FlowJo, Ashland, OR, USA) were used to derive data plots.