Mice were intravenously injected with FITC-conjugated anti-CD45 antibody (BioLegend, San Diego, CA) 3 to 5 min before euthanasia to mark immune cells still present in the vasculature. Following humane euthanasia, mouse lungs were isolated and suspensions of single cells were obtained by enzymatic and mechanical disruption using the gentleMACS dissociator system (Miltenyi Biotec, Auburn, CA) as previously described (54 (link)). Live cells were stained using acridine orange-propidium iodine and counted using a Cellometer K2 cytometer (Nexcelom, Lawrence, MA).
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Lung Cell Isolation and Labeling
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Variable analysis
independent variables
- Intravenous injection of FITC-conjugated anti-CD45 antibody
- Presence of immune cells in the vasculature
- Time between FITC-conjugated anti-CD45 antibody injection and euthanasia (3 to 5 minutes)
- Enzymatic and mechanical disruption of mouse lungs using the gentleMACS dissociator system
- Live cell staining using acridine orange-propidium iodine
- Cell counting using a Cellometer K2 cytometer
- No positive or negative controls were explicitly mentioned in the provided information.
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