MRCs proliferate during inflammation. (A) Flow cytometry gating strategies used to identify MRCs (gp38⁺ MAdCam-1⁺ CD45⁻ CD31⁻ CD21/35⁻) and FDCs (gp38⁺ CD45⁻ CD31⁻ CD21/35⁺) in LN cellular suspensions. WT mice (B) and CD21Cre Ubow⁺⁺ chimeras (C) were subcutaneously injected or not with an emulsion of CFA/OVA. (B) 4, 9, or 20 d later, mice received a single i.p. injection of EdU to label proliferating cells. 1 d later, the percentages of EdU⁺ MRCs and EdU⁺ FDCs present in the peripheral inflamed LNs of WT mice were analyzed by flow cytometry. (C) The percentage of EdU⁺ MRCs (arrowheads) and EdU⁺ FDCs was determined at day 5 by confocal imaging in the inflamed LNs of CD21Cre Ubow⁺⁺ chimeras. Insets display high-magnification views of EdU⁺ MRCs. Bars, 25 µm. Data are representative of 3 experiments (at least 4 mice pooled per group in B and 2 LNs analyzed per mouse in C). (D) WT mice were injected s.c. with CFA/OVA in ears and footpads, followed by BrdU injection (i.p) on day 4. This pulse of BrdU was followed by a chase period of 1 and 3 d. At the end of the chase period, the percentage of BrdU-labeled MRCs and FDCs was determined by flow cytometry. Data are representative of 3 different experiments (5 mice pooled per group). (E) RAG-2°^/° mice were adoptively transferred or not with 6 × 10⁷ WT polyclonal B cells. 1 wk later, mice received a single injection of EdU. The proportion of EdU⁺ cells among the MRCs and FDCs of peripheral LNs were analyzed one day later by flow cytometry. At this stage, no mature FDCs could be recovered from the LNs of both types of mice. Data are representative of 3 different experiments (5 mice pooled per group).