Bronchoalveolar lavage (BAL). Mice were sacrificed at the indicated time by CO2 asphyxiation, and lungs were lavaged five times with 1.0 ml PBS supplemented with 3 mM EDTA each time [11]. Cytospin slides of 2 × 104 BAL Cytochrome c pigeon (88-104) were prepared using a Cytospin 3 centrifuge and stained with HemaStat 3 (Fisher, Pittsburgh, PA). Total cell counts were determined by haemocytometer and differential cell counts were determined by examining 200 cells. The lavage fluid was centrifuged at 2450g for 7 min, and the supernatant collected, filter-sterilized and stored at ?80 °C for cytokine analysis.
Flow cytometric analysis of DCs in the lung. Local recruitment of DCs was evaluated by FACS analysis of lung single-cell suspensions. Briefly, 106 cells were stained with antibodies directed against MHCII (I-Ad), CD40, CD11c, CD80 and CD86 (eBioscience and BioLegend, San Diego, CA), as described previously [12]. Pulmonary DCs were analyzed based on the strategy of Vermaelen and Pauwels [13], and defined as a population of cells expressing high CD11c and low autofluorescence [13].
View User's Journal
Distribution patterns Although both species are considered
 |
