Flow cytometry – cell penetration of 3E10 variants

3E10 antibodies were directly labeled with IVISense 680 NHS Fluorescent Dye (Revvity) in 50 mM carbonate/bicarbonate buffer, pH 8.5 for 2 h at room temperature and purified using Zeba spin desalting columns. K562 cells were pre-treated with inhibitors for 30 min, then treated with inhibitors and 750 nM AlexaFluor 680-labeled antibodies at 37°C, 5% CO₂ for 1 h. Following treatment, cells were washed twice with PBS, trypsinized, and centrifuged at 300 × g for 5 min prior to preparation for flow cytometry as described above. Chemical inhibitors were obtained from the following sources and used at the following concentrations: bafilomycin A1 (Sigma Aldrich), 500 nM; chlorpromazine HCl (Cayman Chemical), 5 μg/mL; dipyridamole (Sigma Aldrich), 50 μM; 5-(N-ethyl-N-isopropyl)-amiloride (Cayman Chemical), 25 μM; filipin III (Cayman Chemical), 1 μg/mL; S-(4-nitrobenzyl)-6-thioinosine (Millipore Sigma), 100 μM. Three biological replicates were performed, and statistical significance was determined using two-way ANOVA.