Biotinylation and Flow Cytometry of Sema5A

Sema5A_TSR3-4 constructs containing a C-terminally fused 3C-Avi-His₆ tag were produced in HEK 293T cells, purified from the secreted medium by Ni-NTA, and site-specifically biotinylated at the AviTag conjugated to their C-terminus in vitro, using a BirA biotin-protein ligase standard reaction kit Avidity LLC (CO, USA, avidity.com). Successful biotinylation was confirmed by Western blot incubated with Streptactin HRP (BioRad) antibody at 1:25,000 dilution (Supplementary Fig. 1j). For each assay sample, 1 × 10⁵ of genetically engineered CHO GS-/- cells^{33 (link),34 (link)} were harvested and washed in PBS before being resuspended in 50 µg/mL of biotinylated WT, R747E/R749E or K734E/R747E/R749E Sema5A_TSR3-4 diluted in PBS with added 1% FBS (assay buffer), gently shaking for 1 h at 4 °C. The cells were then washed with assay buffer before incubation with Alexa Flour 488-streptavidin (1:2000, #S32354, Invitrogen) diluted in assay buffer while gently shaking for 30 min at 4 °C. After wash in assay buffer, the cells were resuspended in assay buffer and subjected to flow cytometry on a SA3800 spectral cell analyzer (SONY), where mean fluorescent intensity for each sample was measured. All experiments were performed a minimum of 3 times using triplicate samples, and mean fluorescent intensity was normalized to CHO WT for all samples.