25 d1.16 pe anti siinfekl h 2kb complex

Poly(ethylene glycol) diacrylate (M_n 700) (PEG₇₀₀DA), 2-aminoethyl methacrylate
hydrochloride (AEM), diphenyl(2,4,6-trimethylbenzoyl)-phosphine oxide
(TPO), thiol-modified CpG 1826 (C6-S–S-C6-tccatgacgttcctgacgtt),
DNase, RNase free sterile water were purchased from Sigma-Aldrich.
Tetraethylene glycol monoacrylate (HP₄A) was synthesized
in house. Cysteine-modified OVA257–264 (CSIINFEKL) was purchased
from Peptide 2.0. Maleimide PEG succinimidyl N-hydroxysuccinimide
(NHS) esters (2k, 5k, and 10k) were purchased from Creative PEG works.
PRINT molds (80 × 320 nm²) were obtained from Liquidia
Technologies. DNA grade NAP-10 columns were purchased from GE Healthcare.
RPMI 1640 medium, penicillin and streptomycin, l-glutamine,
fetal bovine serum (FBS) were all from Life Technologies. BCA protein
assay kit, Pierce LAL Chromogenic Endotoxin Quantitation Kit, RPMI
1640, fetal bovine serum (FBS), and penicillin–streptomycin
(10 000 U/mL) were purchased from Thermo Fisher Scientific.
GM-CSF and IL-4 were purchased from R&D Systems. CD8 T cell isolation
kit and CD11c DC microbeads mouse were purchased from Miltenyi Biotec.
CellTrace Violet Cell Proliferation Kit was obtained from Invitrogen
and CD8-PerCP-eFluor710, CD3-FITC, CD11c-PE, CD11c-APC, and 25-D1.16-PE
(anti-SIINFEKL/H-2K^b complex) antibodies were purchased
from eBioscience. IFN-γ ELISPOT kit was purchased from BD Biosciences.

Day 6 BMDCs (3
× 10⁵ cells) were either untreated or treated with
various formulations or soluble CSIINFEKL peptide at a dose of 5 μg/mL
for 4 h. After 4 h of incubation, cells were either washed with citrate-phosphate
buffer (pH 3.0) for 3 min on ice to strip off the MHC-I–peptide
complex or NP-peptide/H-2K^b complex from the cell surface.
Additionally, cells were reincubated at 37 °C for an additional
20 h postwashes and then stained with CD11c-APC and 25-D1.16-PE (anti-SIINFEKL/H-2K^b complex) antibodies (eBioscience), followed by flow cytometry
analysis on Cyan ADP (Dako).