Localization and kinetics of neutrophil influx in the site of inoculation and in LNs. Mice immunized with OVA/CFA or PBS/CFA were injected with OVA-FITC in the footpads on day 40. (A) Kinetics of NEs (Gr-1, black line) in LNs at different short time periods (10 minutes, 20 minutes, 30 minutes, or 2 hours), evaluated by FACS. The percentage of NEs (Gr-1^high) is also indicated with the gray area corresponding to control background staining. (B) Localization of OVA-FITC⁺ cells in LNs. The left panels show fluorescent microphotographs of LN sections 2 hours (top panels) or 6 hours (bottom panels) after OVA-FITC injection in the footpad of OVA/CFA-immunized mice. The right panels show the corresponding hematoxylin/eosin-stained tissue sections at ×250 and at higher magnification (×1000). Arrows indicate NEs; SS, subcapsular sinuses; C, cortex. (C) Gr-1^high cells (black line) from ipsilateral and contralateral LNs 6 hours after OVA-FITC injection in OVA/CFA-immune mice, processed by FACS. The gray area corresponds to control background staining. (D) Analysis of apoptosis of NEs in LNs or blood obtained 12 or 18 hours after OVA-FITC injection in the footpad of OVA/CFA-immune mice. Cells were stained with FITC-anti–Gr-1 antibody and propidium iodide and then analyzed by flow cytometry. The percentage of cells with hypodiploid DNA in the gate of Gr-1⁺ cells is indicated in each panel. One typical experiment of 3 performed is shown (n = 3-4 mice per group).