Alexa fluor 647 labelled goat anti human igg secondary antibody

ExpiCHO-S cells were seeded at 6 × 10⁶ cells per ml in a volume of 5 ml in a 50-ml bioreactor. The following day, cells were transfected with SARS-CoV-2 S glycoprotein-encoding pcDNA3.1(+) plasmids (BetaCoV/Wuhan-Hu-1/2019, accession number MN908947, Wu-D614; Omicron BA.2, BQ.1.1, XBB.1, XBB.1.5, BN.1 or BA.2-E340A generated by overlap PCR mutagenesis of the Wu-D614 plasmid) harbouring the Δ19 C-terminal truncation. S-encoding plasmids were diluted in cold OptiPRO SFM (Life Technologies, 12309-050), mixed with ExpiFectamine CHO Reagent (Life Technologies, A29130) and added to cells. Transfected cells were then incubated at 37 °C with 8% CO₂ with an orbital shaking speed of 250 rpm (orbital diameter of 25 mm) for 24 to 48 h. Transiently transfected ExpiCHO-S cells were harvested and washed twice in wash buffer (PBS 2% FBS, 2mM EDTA). Cells were counted and distributed into round bottom 96-well plates (Corning, 3799) and incubated with serial dilutions of mAb starting at 10 μg ml⁻¹. Alexa Fluor 647-labelled Goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-606-098) was prepared at 2 μg ml⁻¹ and added onto cells after two washing steps. Cells were then washed twice and resuspended in wash buffer for data acquisition at Ze5 cytometer (Bio-Rad).

Selection of cross-reactive plasma against group 1 and group 2 IAV NAs and the evaluation of the breadth of the best FNI mAbs were performed by binding to NA proteins expressed on the cell surface by flow cytometry. NA expressing ExpiCHO-S or Expi293F cells were harvested, washed twice in FACS buffer (PBS supplemented with 2% FBS and 2 mM of ethylenediaminetetraacetic acid), counted and distributed into 96-well U-bottom plates (Corning). The sera or mAbs were serially diluted and incubated with the cells for 45 min on ice and then washed in FACS buffer. Alexa Fluor 647-labelled goat anti-human IgG secondary antibody (Jackson Immunoresearch) was added at 1 µg ml⁻¹ to the cells, following 20 min of incubation on ice. Cells were then washed with FACS buffer and analysed on a ZE5 Flow Cytometer (Bio-Rad). Data were normalized to the NA-positive cell fractions and analysed with FlowJo software.