Apc ef780 coupled m1 70 anticd11b

A described previously [53 (link)], lungs were harvested, cut into small pieces and incubated for 1 hour at 37°C with a mix of DNAse I (100 μg/ml, Sigma-Aldrich) and collagenase (400 U/ml 1.6 mg/ml, Roche). Lung cells were washed and filtered through a 100 μM filter before being incubated with saturating doses of purified 2.4G2 (anti-mouse Fc receptor, ATCC) in 200 μL PBS 0.2% BSA 0.02% NaN3 (FACS buffer) for 20 minutes at 4°C to prevent antibody binding on the Fc receptor. Various fluorescent mAb combinations in FACS buffer were used to determine cell populations (Table 1). Acquisitions were done on FACScanto II cytofluorometer (Becton Dickinson) with the following mAbs from BD Biosciences: Fluorescein (FITC)-coupled HL3 (anti-CD11c), FITC-coupled 145-2C11 (anti-CD3), APC-coupled RB6-8C5 (anti-GR1), phycoerythrine (PE)-coupled RM4-5 (anti-CD4), PE-coupled E50-2440 (anti-SIGLEC-F), APC-coupled BM8 (anti-F4/80). APC-eF780-coupled M1/70 (antiCD11b) were purchased from eBiosciences and fixable viability dye Aqua (ThermoFisher) was used to gate viable cells. Gating strategies are summarized in S3 Fig.

Harvested lungs were cut into small pieces and incubated for 1 hour at 37 °C with a mix of DNAse I (100 μg/ml, Sigma-Aldrich) and collagenase (1.6 mg/ml, Roche) 400 U/ml. Lung cells were washed and filtered before being incubated with saturating doses of purified 2.4G2 (anti-mouse Fc receptor, ATCC) in 200 μl PBS 0.2% BSA 0.02% NaN3 (FACS buffer) for 20 minutes at 4 °C to prevent antibody binding on the Fc receptor. Various fluorescent mAb combinations in FACS buffer were used to stain 3–5 × 10⁶ cells. Acquisitions were done on FACScanto II cytofluorometer (Becton Dickinson) with the following mAbs from BD Biosciences: Fluorescein (FITC)-coupled HL3 (anti-CD11c), FITC-coupled 145-2C11 (anti-CD3), APC-coupled RB6-8C5 (anti-GR1), phycoérythrine (PE)-coupled RM4-5 (anti-CD4), PE-coupled E50-2440 (anti-SIGLEC-F), APC-coupled BM8 (anti-F4/80). APC-eF780-coupled M1/70 (antiCD11b) were purchased from eBiosciences and fixable viability dye eFluor 506 (eBiosciences) was used to gate viable cells.