For intravenous PE labeling, mice were injected in the tail vein with 2 μg PE anti-CD45.2 (clone 104) and/or 2 μg PE anti-CD45.1 (clone A20) in 100 μl PBS, and euthanized precisely 5 min later. The spleen was quickly harvested and processed through a 70 μm cell strainer in 5 ml ice-cold staining buffer (1× PBS, 2% fetal bovine serum, 0.5 mM EDTA, 0.1% sodium azide, pH 7.3).
For integrin blockade, mice were injected intravenously with 100 μg anti-α4 mAb (clone PS/2) and 100 μg anti-αL mAb (clone M17/4), both from BioXCell. Rat IgG2a (clone 2A3, anti-trinitrophenol) and rat IgG2b (clone LTF-2, anti-keyhole limpet hemocyanin), also from BioXCell, were used as isotype controls.
Mice were treated with various doses of FTY720 (Cayman Chemical). For marginal zone B cell shuttling experiments, mice were treated with 1 mg/kg FTY720 intraperitoneally the night before sacrifice. For immunofluorescence experiments, mice were treated with either 1 mg/kg intravenously 6 h prior to sacrifice or 3 mg/kg intraperitoneally 18 h before sacrifice followed by 2 mg/kg intravenously 6 h before sacrifice.
For integrin blockade, mice were injected intravenously with 100 μg anti-α4 mAb (clone PS/2) and 100 μg anti-αL mAb (clone M17/4), both from BioXCell. Rat IgG2a (clone 2A3, anti-trinitrophenol) and rat IgG2b (clone LTF-2, anti-keyhole limpet hemocyanin), also from BioXCell, were used as isotype controls.
Mice were treated with various doses of FTY720 (Cayman Chemical). For marginal zone B cell shuttling experiments, mice were treated with 1 mg/kg FTY720 intraperitoneally the night before sacrifice. For immunofluorescence experiments, mice were treated with either 1 mg/kg intravenously 6 h prior to sacrifice or 3 mg/kg intraperitoneally 18 h before sacrifice followed by 2 mg/kg intravenously 6 h before sacrifice.