The presence of membrane proteins on the surface of BNPs was determined by Western blotting as published [13 (link)]. Membrane-coated PLGA NPs were purified by centrifugation at 9600×g for 10 min. Whole T-cell-enriched culture extract, membrane fraction, and membranes isolated from coated PLGA NPs by centrifugation at 9,600×g for 10 min (30 µg protein) were subjected to electrophoresis and transferred onto a nitrocellulose membrane. Membranes were incubated overnight at 4 °C with primary antibodies against GAPDH (1E6D9, Proteintech) or Na+/K+-ATPase α1 (C464.6, Santa Cruz Biotechnology) (1:1,000 dilution), and the secondary antibody (Cell Signaling) (1:2,000 dilution) for 1 h at room temperature. The chemiluminescence signal was obtained with an ImageQuant LAS 4000 (GE Healthcare).
The surface expression of IC receptors on NExT was determined by flow cytometry (FACSVerse flow cytometer, BD Biosciences) with no threshold on the forward scatter to detect the nanoparticles as previously reported [6 (link), 36 (link)]. Briefly, NExT were incubated for 15 min at room temperature with anti-PD1, anti-LAG3, and anti-TIM3 antibodies, or their corresponding isotype, as described above. The results were analyzed by FlowJo.
The surface expression of IC receptors on NExT was determined by flow cytometry (FACSVerse flow cytometer, BD Biosciences) with no threshold on the forward scatter to detect the nanoparticles as previously reported [6 (link), 36 (link)]. Briefly, NExT were incubated for 15 min at room temperature with anti-PD1, anti-LAG3, and anti-TIM3 antibodies, or their corresponding isotype, as described above. The results were analyzed by FlowJo.
Free full text: Click here
