Oligreen

2mg of MSRs were loaded with either 2ug of GM-CSF, 100ug of CpG, or 10ug of Rhodamine-PEI (L25K). The individual vaccine components were then resuspended in 1ml of release media, which is composed of RPMI (Sigma-Aldrich) supplemented with 1% penicillin/ streptomycin and 10% heat-inactivated FBS (Sigma-Aldrich), in low-binding Eppendorf tubes. Release study was then performed in 37 °C under gentle shaking. Periodically, supernatant containing the released component was collected by pelleting the particles at 2000g for 10 min. The release media was subsequently replaced. For analysis, GM-CSF content was determined using ELISA (R&D). CpG content was detected using OliGreen (ThermoFisher)^{13 (link)}.

CABV complexation of 25-mer phosphorothioate gapmer oligonucleotides was determined using an Oligreen (Thermo Fisher Scientific, Wilmington, DE, USA) unsymmetrical cyanine dye-exclusion assay. Binding was measured by exclusion of dye from oligonucleotide binding through triplicate testing of serial doses of CABVs with 2 μg/mL gapmer ASO solutions placed in black 96-well optical bottom microplates (Nunc; Thermo Fischer Scientific, Wilmington, DE, USA). Each CAB was suspended in DMSO to a concentration of 20 mg/mL, preparing 1× and 10× working solutions of CABVs seeded in dilute water. Each solution was sonicated on ice for 30-minutes with a high-intensity probe using 30 sec on/off cycles at 25% amplitude with a power setting of 6W.
A working dilution was then prepared with 150 μL Oligreen added to 150 μL 200× TE and 15 μL of nfH20. Scrambled 25-mer gapmer stocks (1 mM) were suspended in 46.3 μL of 10 mM Tris-HCL (ph 8.0), preparing 50 μM gapmer working dilutions; 5 μL of this solution was added to separate triplicate well sets in 3 × 96 well plates, with the addition of both 1× and 10× CABV solutions added to gapmer solutions to accomplish a stepwise mixing of CABVs. Following a 20-minute incubation period to stabilize the fluorescence signal, fluorescent readings (485 nm excitation, 520 nm emission; 20 nm bandwidth) were recorded and complexation curves were created.