Supplementary MaterialsSupplementary figures and furniture: Physique S1 shows the impact of MSA on T cell activation; Physique S2 shows accumulation dynamics of the MSA/ABD-iTEP-pOVA complex and ABD-iTEP-pOVA in cytosol and vesicles; Physique S3 includes fluorescent images of the MSA/ABD-iTEP-pOVA complex and ABD-iTEP-pOVA in cytosol and vesicles; Table S1 summarizes pharmacokinetic parameters of ABD-iTEP and iTEP. this fusion protein, termed ABD-iTEP, and mouse serum albumin (MSA). Next, we evaluated the accumulation of ABD-iTEP in LNs and dendritic cells (DCs) in the LNs. We also analyzed antigen presentation and T cell activation of vaccines that were delivered by ABD-iTEP and investigated possible underlying mechanisms of the presentation and activation results. Last, we measured CTL responses induced by ABD-iTEP-delivered vaccines and immune assays experienced an endotoxin level 0.25 EU/mg. Native polyacrylamide gel electrophoresis (PAGE) 20 M MSA (Sigma-Aldrich) was incubated with ABD-iTEP of different concentrations including 10, 20, 40 and 80 M overnight at 4 C. 5 L of each sample was mixed with 5 L sample buffer and loaded into each well of the gel. The electrophoresis was run at 150 V for 2 h. The gel was then stained with Coomassie Amazing Blue R-250 dye. The image of the gel was taken using the FluorChem FC2 imaging system (Alpha Innotech). Size exclusion chromatography (SEC) SEC was conducted on an Agilent 1260 SEL10 infinity liquid chromatography system (Agilent Technologies) equipped with an Agilent ZORBAX GF-450 size exclusion column (diameter: 9.4 mm, length: 250 mm, particle diameter: 6.0 m). 60 M ABD-iTEP was incubated with 30 M MSA immediately at 4 C. 100 L sample was loaded for analysis. Samples were eluted with PBS at a circulation rate of 1 1 mL/min. Spectra were monitored by absorption at 280 nm. Surface plasmon resonance (SPR) assay The SPR measurement of binding affinities were measured by a MASS-1 SPR instrument (Sierra Sensors). Following a standard LCL-161 manufacturer amine-coupling process, MSA was immobilized on a SPR affinity sensor (High Capacity Amine, Sierra Sensors). iTEP, ABD-iTEP, iTEP-pOVA, and ABD-iTEP-pOVA were diluted in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20 and 0.2 mg/mL ovalbumin) to 10 nM. The samples were injected at a constant flow rate of 25 L/min for 3 min, followed by injection of running buffer for 30 min. After each injection cycle, the surface was regenerated with two injections of 25 L of 10 mM HCl. After subtracting reference surface and buffer injection, the data were fitted to the one-to-one Langmuir binding model with mass transport limitations to calculate the association rate constant (dendritic cell accumulation assay C57BL/6 mice were subcutaneously injected with 1.5 nmol of Alexa Fluor 488 labelled iTEP or ABD-iTEP at each side of the base of tail. Inguinal and axillary LNs were isolated 6 h after injection. LNs were pooled and mechanically homogenized to prepare a single-cell suspension by passing through a 40 m nylon mesh. Cells were then washed and stained with PE anti-mouse CD11c (Clone: N418, Biolegend) and 4,6-diamidino-2-phenylindole (DAPI). Circulation cytometric analysis was conducted using a BD Canto (BD Biosciences). The results were analyzed by FlowJo software. dendritic cell uptake assay This assay was conducted using a previous method with some modifications 32. DC2.4 cells were cultured with medium containing 5 M fluorescein-labelled polypeptides for 2 h. The medium was then removed and cells were washed. Trypan blue was added to quench extracellular fluorescence and cells were washed another two times. 0.2% Triton X-100 was added to lyse the cells and release the endocytosed polypeptides. The amount of endocytosed polypeptide was detected by measuring the fluorescence intensity. Antigen presentation assay DC2.4 cells were cultured in medium containing vaccines at a concentration of 5 M for 16 h. The cells were then collected and stained with PE anti-mouse H-2Kb/SIINFEKL antibody (Clone: 25-D1.16, Biolegend) LCL-161 manufacturer and DAPI. SIINFEKL (pOVA) presented on the cellular surface was quantified by flow cytometric analysis. The results are presented as median fluorescence intensity (MFI) relative to the MFI of untreated DC2.4 cells. B3Z cell LCL-161 manufacturer activation assay B3Z cell is a CD8+ T-cell hybridoma 33. Upon recognition of H-2Kb/SIINFEKL complex, B3Z cells will be LCL-161 manufacturer activated to produce -galactosidase, which can hydrolyze the.