Ab157723

Standard commercial ELISA kits were used for the quantification of Tnf (BMS607-3, Thermofisher, Scoresby, VIC, Australia) and Saa (ab157723, Abcam, Cambridge, MA, USA) in serum. The concentration levels of cytokines were determined by standard curves for each cytokine using a plate reader at an absorbance of 450 nm (TECAN, Port Melbourne, VIC, Australia) and reported as pg/mL for Tnf and ng/mL for Saa.

Bone marrow cells were harvested and immediately cryopreserved on-site at UNC-CH according to prior published methods [28 (link)]. Cryopreserved cells were transported on dry ice to the University of Virginia and stored at -80 °C. Cells were thawed and cultured in RPMI with 10% FBS supplemented on days 0 and 3 with GM-CSF (10 ng/mL; Peprotech), and harvested on day 6. For in vitro experiments, 2.5x10⁵ cells were plated per well of a round-bottom 96-well dish, and treated with lipopolysaccharide (Ultra-pure LPS-EK, Invitrogen, 1mcg/mL) for 24h. Cytokines in the supernatants were determined by ELISA (IL-23, IL-10 R&D Systems) and Luminex (DropArray 96 plate for Bio-PlexTM, Curiox). Cells from at least three mice per group, in duplicate, were utilized. Results were analyzed using One Way ANOVA or, Kruskal-Wallis test with Turkey or Dunn’s post -test, based on Shapiro–Wilk test for normality, in Graphpad Prism 7 software (San Diego, CA).
Serum SAA was measured by ELISA (Abcam, Cambridge, England, ab157723).