Label it nucleic acid labeling kits cy5

TLR9 agonist CpG oligonucleotide (5’T*C*C*A*T*G*A*C*G*T*T*C*C*T*-G*A*C*G*T*T 3’) was synthesized by IDT with a phosphorothioate backbone. Label- IT nucleic acid labeling kits (Cy5) were purchased from Mirus Bio LLC. Gelatin from cold water fish skin 40-50% in H2O was purchased from Sigma-Aldrich. A Sylgard silicone elastomer kit was obtained from Dow Corning. The HEK TLR reporter cell line HEK-Blue mTLR9 cell line was purchased from InvivoGen (SanDiego).

SIINFEKLR₉ (SIINR₉) was synthesized by Genscript with >98 % purity, with a FITC label on the N-terminus. Low molecular weight polyinosinic-polycytidylic acid (polyIC) was purchased from Invivogen. iPEMs were synthesized as previously described with the exception that all cargo and wash solutions were in DI water. [2 (link)] After the final layer, the calcium carbonate core was removed by resuspending the iPEMs in 1 mL 0.1 M ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich), adjusted to pH 4 with 1 M HCl and 1 M NaOH solutions. Capsules were then centrifuged at 2000g for 2 min and washed with 100 μL 1X PBS. iPEMs were collected by centrifuging at 1000g for 1 min and then resuspended in 100 μL PBS. In some studies, iPEM capsules were formed from FITC SIINR₉ and Cy5 labeled polyIC following the same protocol as above. Label-IT nucleic acid labeling kits (Cy5) were purchased from Mirus Bio LLC. PolyIC was labeled according to the Mirus Bio instructions.