2.2. Enrichment of stable cell pools by FACS
With the aim of selecting high-producer subpopulations, at six weeks
post-transfection a total of 200 × 106 cells were
stained using 4G2 mAb (biotinylated in house using EZ-Link biotinylation
kit #21425, Thermofisher, USA) and Streptavidin-phycoerythrin (PE)
(#S866, Thermofisher, USA). Cells were then sorted by FACS
(fluorescence-activated cell sorting) in order to collect cells showing
higher fluorescent signal, as described previously (Alvim et al., 2019).
After these sorted cells (named cell pool 1) recovered viability, a
second round of sorting was performed on week 14 post-transfection and
high-fluorescence cells were collected and named cell pool 2. Documented
cell banks of both cell pools were cryopreserved. Kinetic studies were
carried out to evaluate cell growth, metabolism and VLP production.