Characterization of stromal cell primed by TNF/LT vs IL-4. (A) CXCL12 was quantified at RNA (RT-qPCR; left) and protein (ELISA; right) levels in ADSCs treated by TNF/LT or IL-4 for 3 days. In RT-qPCR experiments, an arbitrary value of 1 was assigned to untreated cells (UNT). Results represent the mean ± standard deviation (SD) from 7 experiments. (B) ADSCs were stimulated by TNF/LT, IL-4, or left untreated (UNT) for 3 days before quantification of VCAM1, TGM2, and PDPN expression by RT-qPCR (n = 7). The arbitrary value of 1 was assigned to UNT cells. In addition, VCAM-1/CD106 expression was evaluated by flow cytometry (n = 5) and the ratio of mean fluorescence intensity (RMFI) was determined by comparison with an appropriate control isotype. (C) ADSCs were cultured for 3 days with TNF/LT, IL-4, or left untreated (UNT) before fixation and staining with transglutaminase and podoplanin. Nuclei were counterstained with SytoxBlue (white). Scale bars, 50 µm. (D) ADSCs were cocultured for 2 days with purified FL B cells expressing detectable (TNF^hi; n = 6) or undetectable (TNF^lo; n = 7) amounts of TNF as measured by ELISA, before sorting of CD45^–CD105⁺DAPI^– viable stromal cells and CXCL12 quantification by RT-qPCR. The arbitrary value of 1 was assigned to ADSCs cultured alone (UNT). (E) ADSCs were cocultured for 2 days with purified FL-T_FH cells before sorting of CD45^–CD105⁺ DAPI^– viable stromal cells and CXCL12 quantification by RT-qPCR. Results represent the mean ± SD from 4 experiments. **P < .01; *P < .05.