Lungs were perfused with cold PBS via right ventricle, minced and digested with collagenase IV (5 mg/ml) and DNase I [24 (link)]. Lung digests were passed through 70 μ filter, cells were harvested by centrifugation, and treated with RBC lysis buffer. Cells were then washed and suspended in PBS.
To determine differential cell counts, an aliquot of cell suspension equivalent to 1 x 106 cells was labeled with antimouse CD45 antibody conjugated with magnetic microbeads (Miltenyi Biotec Inc, Auburn, CA) to isolate CD45 positive cells. Cytospins of CD45 positive cells were prepared, stained with DiffQucik and number of macrophages, neutrophils and T cells were determined.
For flow cytometry analysis, cells obtained after RBC lysis were incubated with Zombie UV™ (BioLegend) to label dead cells and stained with fluorescence-labeled Abs against surface markers of leukocytes, such as CD45, CD11c, CD11b, F4/80, CD3e, CD8 and CD4. Appropriate isotype-matched controls and fluorescence minus one (FMO) were used in all experiments. All antibodies were purchased from BioLegend (San Diego, CA). Cells were fixed and analyzed in BD LSR II Flow cytometerI (BD Biosciences) and data was analyzed using FlowJO version 10 (Tree Star, Ashland, OR).
To determine differential cell counts, an aliquot of cell suspension equivalent to 1 x 106 cells was labeled with antimouse CD45 antibody conjugated with magnetic microbeads (Miltenyi Biotec Inc, Auburn, CA) to isolate CD45 positive cells. Cytospins of CD45 positive cells were prepared, stained with DiffQucik and number of macrophages, neutrophils and T cells were determined.
For flow cytometry analysis, cells obtained after RBC lysis were incubated with Zombie UV™ (BioLegend) to label dead cells and stained with fluorescence-labeled Abs against surface markers of leukocytes, such as CD45, CD11c, CD11b, F4/80, CD3e, CD8 and CD4. Appropriate isotype-matched controls and fluorescence minus one (FMO) were used in all experiments. All antibodies were purchased from BioLegend (San Diego, CA). Cells were fixed and analyzed in BD LSR II Flow cytometerI (BD Biosciences) and data was analyzed using FlowJO version 10 (Tree Star, Ashland, OR).
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