Lectin Binding Evaluation by Flow Cytometry

Lectin binding was evaluated by flow cytometry as described previously^{48 (link)}. The following reagents were used at the final concentrations indicated: allophycocyanin-streptavidin (APC-Strep) (Life Sciences, S-868), 5 ug/mL), LEA-biotin (Vector labs B-1175, 1 μg/mL), L-PHA rhodamine (Vector labs RL-1112, 20 μg/mL). Empty vector (EV) control and LKB1 WT expressing H460 and H2122 cells, and doxycycline-inducible GFPT2 KO H460 and H157 cells were harvested by centrifugation, washed twice with DPBS, then resuspended in DPBS at 2.0 × 10⁶ cells/ml. Then, 200 μL of cell suspension was transferred to a V-bottom 96-well plate and pelleted by centrifugation at 600g for 5 minutes. The cell pellets were incubated with 100 μL of lectin diluted in DPBS for 60 min at 4 °C (LEA incubation was 30 min), then washed with DPBS three times. When a secondary detection reagent was used, cells were incubated with 5 μg/mL APC-Strep in DPBS for 45 min at 4 °C. Fluorescence was analyzed by flow cytometry on a FACS Aria II SORP with dual lasers at 488 nm and 635 nm. Plots show the mean fluorescence intensity (MFI), typically for 10,000 cells.