Supramolecular Organization Predicts Protein Nanoparticle Delivery to Neutrophils for Acute Lung Inflammation Diagnosis and Treatment

Jacob W. Myerson; Priyal N. Patel; Nahal Habibi; Landis R. Walsh; Yi-Wei Lee; David C. Luther; Laura T. Ferguson; Michael H. Zaleski; Marco E. Zamora; Oscar A. Marcos-Contreras; Patrick M. Glassman; Ian Johnston; Elizabeth D. Hood; Tea Shuvaeva; Jason V. Gregory; Raisa Y. Kiseleva; Jia Nong; Kathryn M. Rubey; Colin F. Greineder; Samir Mitragotri; George S. Worthen; Vincent M. Rotello; Joerg Lahann; Vladimir R. Muzykantov; Jacob S. Brenner

For analysis of intravascular leukocyte populations in naïve and inflamed lungs, mice received an intravenous injection of FITC-conjugated anti-CD45 antibody five minutes prior to sacrifice and preparation of single cell suspensions as described above. Populations of intravascular vs. extravascular leukocytes were assessed by subsequent stain of fixed cell suspensions with PerCP-conjugated anti-CD45 antibody and/or APCconjugated clone 1A8 anti-Ly6G antibody. To accomplish staining of fixed cells, 100 µL aliquots of the cell suspensions described above were pelleted at 400xg for 5 minutes, then resuspended in labeled antibody diluted in FACS buffer (1:150 dilution for APCconjugated anti-Ly6G antibody and 1:500 dilution for PerCP-conjugated anti-CD45 antibody). Samples were incubated with staining antibodies for 20 minutes at room temperature in the dark, diluted with 1 mL of FACS buffer, and pelleted at 400xg for 5 minutes. Stained pellets were resuspended in 200 µL of FACS buffer prior to immediate flow cytometric analysis on a BD Accuri flow cytometer. All flow cytometry data was gated to remove debris and exclude doublets. Control samples with no stain, obtained from naïve and IV-LPS-injured mice, established gates for negative/positive staining with FITC, PerCP, and APC. Single stain controls allowed automatic generation of compensation matrices in FCS Express software. Comparison of PerCP anti-CD45 signal with FITC anti-CD45 signal indicated intravascular vs. extravascular leukocytes. Comparison of APC anti-Ly6G signal with FITC anti-CD45 signal indicated intravascular vs. extravascular neutrophils, with PerCP and APC co-staining verifying identification of cells as neutrophils.