Rat anti mouse cd45 apc cy7

Auricular LNs were dissected from mice with and without skin inflammation on day 6 and digested as previously described [15 (link)]. The capsule was disrupted with two 25G needles and the LNs incubated for 20 min at 37 °C in digestion medium consisting of DMEM with 2% FCS, 1.2 mM CaCl₂, 40 µg/mL DNase I, and 1 mg/mL Collagenase IV (17104-019, Gibco). After incubation, the supernatant was removed and the LN fragments washed once with DMEM. The fragments containing the LN stromal cells were then incubated again at 37 °C for 15 min in 750 µL digestion medium containing 3.5 mg/mL Collagenase IV. The remaining fragments were then disrupted by pipetting up and down 2 × 100 times, with addition of 5 mM EDTA. The cell suspension was filtered through a 40 µm cell strainer, washed and treated with Fc-block (rat anti-mouse CD16/32, 101302, BioLegend) in FACS buffer (DPBS with 1% FBS, 2 mM EDTA and 0.02% NaN₃) for 20 min. Cells were stained with rat anti-mouse CD45 APC-Cy7 (103116, BioLegend), rat anti-mouse CD31 APC (551262, BD Biosciences), hamster anti-mouse podoplanin PE (25-5381-82, eBioscience), rat anti-mouse CD200 PE-Dazzle594 (123820, BioLegend) and ZombieNIR diluted in PBS. The cells were then fixed with the Anti-Mouse/Rat Foxp3 PE staining set (72-5775-40, eBioscience) according to the kit’s instructions, and data were acquired on a BD LSRFortessa.

ECs defined as CD31-positive, CD45-negative cells were isolated from liver (± 6 mice for each experiment) and sorted according to previously published protocols [6 (link)]. For fetal samples, incubation with shaking step was omitted. Cell surface staining was performed using the following antibodies: rat anti-mouse CD31 BV421 (Biolegend, San Diego, USA), rat anti-mouse CD45 FITC (eBiosciences, San Diego, USA), rat anti-mouse CD45 APC-Cy7 (Biolegend), mouse anti-mouse CD157 APC (Biolegend), and rat anti-mouse CD200 PE (Biolegend), all at 1:200 dilution. To exclude dead cells, propidium iodide (PI; Sigma-Aldrich Japan) was added before analysis and sorting on a BD FACSAria (BD Biosciences, San Diego, USA). Flow cytometry data quantification was performed using FlowJo v10 (BD Biosciences).