Manager 5

3–5 mice were used per treatment group for all assays in two independent experiments. Mice were sacrificed at indicated times and whole blood was drawn via cardiac puncture. Serum was isolated and used in custom Bioplex cytokine assays (Bio-Rad Laboratories, Hercules, CA). Plates were read using the Bioplex 200 plate reader and analyzed with Bio-Plex Manager 5.0 software.

Total RNA was extracted using RNeasy mini kits (Qiagen, Valencia, CA) as described in the manufacturer's protocol. Real time reverse transcriptase polymerase chain reactions (RT-PCR) using Bio-Rad iCycler was performed to determine the mRNA expression levels. The reaction conditions included reverse transcription at 50°C for 30 min, denaturation at 95°C for 15 min, followed by amplification for 45 cycles (95°C for 15 sec, 54°C for 30 sec, and 76°C for 15 sec). The CCL5 primers are 5′ ACC AGT GGC AAG TGC TCC A-3′ for forward and 5′ ACC CAT TTC TTC TCT GGG TTG GCA-3′ for reverse (Integrated DNA Technology, Coralville, IA). Separate hypoxanthine-guanine phosphoribosyl-transferase (HPRT - house-keeping gene) amplification was used to normalize gene expression. The data was analyzed using the equation 2^−ΔΔCT method. The primer sequences and PCR conditions for p38 and Akt isoforms used in this study and the annealing temperatures were based on our previous study30 (link).
Cell culture supernatants were collected and the cytokine protein concentrations were analyzed by a multi-cytokine bead assay system (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. The protein expression was measured using Bio-Plex Manager 5.0 software and 5PL standard curve.

Levels of sixteen immune checkpoint proteins (BTLA, CD27, CD28, CD40, CD80/B7-1, CD86/B7-2, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, TIM-3, and TLR2) were determined in CVL samples using the MILLIPLEX MAP® Human Immuno-Oncology Checkpoint Protein Magnetic Bead Panel (Millipore, Billerica, MA) in accordance with the manufacturer’s protocol. Data were collected with a Bio-Plex® 200 instrument and analyzed using Manager 5.0 software (Bio-Rad, Hercules, CA). A four-parameter logistic regression curve fit was used to determine the concentration. All samples were assayed in duplicate. The concentration values below the detection limit were substituted with 0.5 of the minimum detectable concentration provided in manufacturer’s instructions. The natural logarithm (ln) transformation was applied to normalize the data.

Cell culture supernatants from 3-D cervical cell models infected with M. mulieris UPII-28I and Eggerthella sp. MVA1 were collected from three independent experiments. The levels of five cytokines: (interleukin (IL)-1α, IL-1β, IL-1RA, IL-6, tumor necrosis factor-α (TNF)-α), seven chemokines: fractalkine, IL-8, interferon γ-induced protein-10 (IP-10), monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein-1β (MIP-1β), regulation on activation, normal T-cell expressed and secreted (RANTES) and three growth factors: platelet derived growth factor-AA (PDGF-AA), transforming growth factor-α (TGF-α), vascular endothelial growth factor (VEGF) were measured using customized MILLIPLEX^® multianalyte profiling (MAP) Human Cytokine/Chemokine Panel 1 array (Millipore) and compared to PBS mock infections. Data was collected using a Bio-Plex® 200 (Bio-Rad) platform and evaluated using Manager (5.0) software (Bio-Rad). A five-parameter logistic regression curve fit was used to determine the concentration. All samples were analyzed in biological triplicate, each containing two technical replicates.

Levels of 28 soluble proteins, including cytokines (IL-1α, IL-1β, IL-6, MIF, TNFα, TRAIL), chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL20/MIP-3α, CXCL8/IL-8/, CXCL10/IP-10, growth factors (TGF-α, VEGF), MMPs (MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, MMP-10), mucins (MUC1/CA15-3, MUC16/CA125), CEA, the death receptor sFas and its ligand sFasL, the heat shock protein HSP70, and the cytokeratin fragment CYFRA 21-1, were determined in cell culture supernatants using customized cytometric magnetic bead arrays based on Luminex® xMAP® technology: MILLIPLEX® MAP Human Cytokine/Chemokine Panel 1, Th 17 Panel, Circulating Cancer Biomarker Panel 1, Sepsis Panel 2, and MMP Panel 2 (Millipore, Billerica, MA, USA). The assays were performed in accordance with the manufacturer’s protocols. Data were collected with a Bio-Plex® 200 instrument and analyzed using the Manager 5.0 software (Bio-Rad, Hercules, CA, USA). A five-parameter logistic regression curve fit was used to determine the concentration. All samples were assayed in duplicate.