Lipoteichoic acid lta from streptococcus faecalis

Whole blood from tubes containing lithium heparin were diluted 1:2 with RPMI 1,640 culture medium containing 200 U of penicillin/mL and 200 mg of streptomycin/mL, transferred to 24-well plates, and stimulated with lipoteichoic acid (LTA) from Streptococcus faecalis (final concentration 1 μg/mL, Sigma Aldrich), lipopolysaccharide (LPS) from E. coli 0127:B8 (final concentration, 100 ng/mL, Sigma Aldrich), or phosphate-buffered solution (PBS) as a negative control. Samples were incubated in the dark at 37°C in 5% CO₂ for 24 h. The samples were then centrifuged (400 g for 7 min) and supernatant collected and stored at −80°C for batch analysis. For analysis, samples were thawed, and then tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-8, and monocyte chemoattractant protein (MCP)-1 were quantified with a canine cytokine-specific multiplex bead-based assay (Milliplex MAP, EMD Millipore Corp.) as described elsewhere (16 (link)). The median fluorescence intensity and cytokine concentration in each sample was measured in duplicate with appropriate controls and associated data analysis software (Milliplex Analyst version 5.1, EMD Millipore Corp.). The lower limit of detection for TNF-α, IL-6, IL-10, GM-CSF, IL-2, and MCP-1 was 48.8 ρg/mL. The lower limit of detection for IL-8 was 195.3 ρg/mL.

After incubation with calcitriol or control, blood-RPMI mixture was transferred to 96-well plates and stimulated with lipopolysaccharide (LPS) from Escherichia coli O127:B8 (final concentration, 100 ng/mL, Sigma Aldrich, St Louis, MO), lipoteichoic acid (LTA) from Streptococcus faecalis (final concentration, 1 μg/mL, Sigma Aldrich, St Louis, MO), or phosphate-buffered saline (PBS) control substance. Plates were incubated for 24 h at 37 °C in 5% CO₂ in the dark. Following incubation, plates were centrifuged (400 X g for 7 min) at 21 °C as previously described [11 (link)]. The supernatant was collected and stored at − 80 °C for batch analysis. For analysis, samples were thawed, and then TNF-α, IL-6, and IL-10 were measured in supernatant with a canine cytokine-specific multiplex bead-based assay (Milliplex MAP canine cytokine-chemokine panel, EMD Millipore Corp, Billerica, MA) [11 (link)]. The median fluorescence intensity and cytokine concentration in each sample was measured in duplicate with appropriate controls and associated data analysis software (Milliplex Analyst version 5.1, EMD Millipore Corp, Billerica, MA). The lower limit of detection for TNF-α, IL-10 was 195 ρg/mL and IL-6 was 48.8 ρg/mL.

After incubation with calcitriol or ethanol for 24 h, samples from the conical tubes were transferred to 96-well plates and stimulated with lipopolysaccharide (LPS) from Escherichia coli O127:B8 (final concentration, 100 ng/mL, Sigma Aldrich, St Louis, MO, USA), lipoteichoic acid (LTA) from Streptococcus faecalis (final concentration, 1 µg/mL, Sigma-Aldrich, St Louis, MO, USA), or a phosphate-buffered saline (PBS) control substance. Plates were incubated for 24 h at 37 °C in 5% CO₂ in the dark. Following incubation, plates were centrifuged (400× g for 7 min) at 21 °C as previously described [40 (link)]. The supernatant was collected and stored at −80° C for batch analysis. For the analysis, samples were thawed, and then TNF-α, IL-6, IL-8, and IL-10 were measured in supernatant with a previously validated canine cytokine-specific multiplex bead-based assay (Milliplex MAP canine cytokine–chemokine panel, EMD Millipore Corp, Billerica, MA, USA). The median fluorescence intensity and cytokine concentration in each sample was measured in duplicate with the appropriate controls and associated data analysis software (Milliplex Analyst version 5.1, EMD Millipore Corp, Billerica, MA, USA). The lower limit of detection for TNF-α, IL-10, and IL-6 was 48.8 pg/mL, and for IL-8, it was 195 pg/mL.