Anti flag m2 fitc

Mammalian cell display vector harboring Fc libraries was transfected into HEK293T cells, together with pMDLg/pRRE, pRSV-Rev and pCMV-VSV-G packaging plasmids, using polyethylenimine (PEI), to produce lentivirus library. The lentivirus was concentrated with Lentivirus Concentration Reagent (BIOMIGA), and the titer was measured with Lenti-X™ p24 Rapid Titer Kit (Takara Bio Inc.).
1×10⁷ HEK293T cells were infected by lentivirus library at low MOI, followed by 48 h of culture to display Fc variants on the cell surface. The cells were then detached by StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific), resuspended in 1 mL blocking buffer (PBS supplemented with 5% FBS (V/V), 1% glucose (W/V), 1 mM Na₂EDTA, 1 mM HEPES) and incubated for 30 min at 4 °C with gentle agitation. Then the cells were incubated with 250 nM (125 nM for the 2^nd and 3^rd rounds of sorting) biotinylated FcγRs (bio-FcγRs) in blocking buffer at 4 °C for 30 min. After washing three times with ice-cold FACS buffer (PBS supplemented with 1% glucose (W/V), 1 mM Na₂EDTA, 1 mM HEPES), the cells were resuspended in 1 mL of blocking buffer containing streptavidin-PE (1:1000 dilution, Thermo Fishier Scientific) and ANTI-FLAG® M2-FITC (1:200 dilution, SIGMA) and incubated at 4 °C for 30 min. The cells were finally washed three times and resuspended in ice-cold FACS buffer for sorting (FACSAria III, BD).

The N2A cells were plated on coverslips precoated with poly L-lysine (Sigma, P4707). Twenty-four hours after transfection, N2A cells were differentiated by 0.1% FBS and 2 mM retinoic acid for 24 h. Differentiated N2A cells were washed with PBS, fixed by 4% PFA, permeabilized with 0.1% Triton X-100 in PBS, blocked with 10% BSA in PBS, incubated with Anti-Flag M2 FITC (Sigma, F4049), and visualized on confocal microscope (Nikon A1R). For cell counting, we replicated n = 4 independent transfection and took ten nonoverlapped images for each transfection. An average of 250 cells were counted for each transfection.