Quantification of Cytokine Release in Gingival Infection

Culture supernatants were harvested after P. gingivalis infection (100 MOI) and analyzed using Human IL-6/IL-8 ELISA Ready-SET-Go!, 2nd Generation Kit (Invitrogen, Thermo Fisher Scientific) following users’ manual (7 (link)). Briefly, 96-well plates were coated with Capture antibody in coating buffer and incubate at 4°C overnight. After washing with wash buffer (0.05% Tween in PBS) and blocking with 1× Diluent, the supernatants were then added to the wells and incubated for 2 h in room temperature. Then Detection antibody was added and followed by Avidin incubation. The 96-well plates were washed with wash buffer as directed after each incubation. TMB buffer was added to the wells following stop solution addition. The plates were read at 450 nm with Multiskan MCC (Thermo Fisher Scientific). For qRT-PCR, RNAs in TIGKs were prepared with RNeasy Plus mini kit (Qiagen) using Qiacube (Qiagen) and processed following published protocols (7 (link)). cDNAs were synthesized from 800 ng of total RNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and were then used as templates for qRT-PCR with SYBR Green Master Mix (SABiosciences) on Applied Biosystems 7500. The primers used in qRT-PCR are listed in Table 1.