Three experimental strategies demonstrated CD44 was essential for inhibition by TSG-6. (A) Immunocytochemistry assay for expression of CD44 in clones of NF-κB reporter cells stably transfected with control plasmid (HEK-hTLR2-pcDNA) or plasmid containing complementary DNA for CD44 (HEK-hTLR2-CD44). (B) Assays for NF-κB signaling in the reporter cells expressing secreted alkaline phosphatase (SEAP). HEK-hTLR2-pcDNA and HEK-hTLR2-CD44 were incubated for 7 hours with zymosan with or without rhTSG-6. Values are mean ± SD (n = 3; **P < .001). (C) Murine macrophages pre-incubated for 15 minutes with control IgG (rat IgG_2a) or blocking antibody for CD44 (CD44 KM81) and then incubated for 4 hours with zymosan and with or without activated hMSCs or TSG-6 (conditions as in Figure 2E). (D) Real-time RT-PCR assays with mouse-specific primers on resident macrophages isolated from wild-type mice (CD44^+/+) and transgenic mice (CD44^−/−) after injection of zymosan followed by injection of HBSS (n = 9 for CD44^+/+ and n = 5 for CD44^−/−), 1.6 × 10⁶ hMSCs (n = 5 for CD44^+/+ and n = 3 for CD44^−/−) or 30 μg of rhTSG-6 (n = 3 for CD44^+/+ and CD44^−/−). Values are mean ± SD (*P < .05; **P < .005).