Ly 6b 2

For murine samples, lungs were fixed in 10% formalin, then processed and embedded in paraffin by standard procedures. Sections (5 μM) were stained with hematoxylin and eosin (H/E) and examined by a pathologist blinded to the experimental groups. For immunohistochemistry, formalin-fixed paraffin-embedded lungs were cut into 4 μM sections. F4/80 (Cell Signaling, D2SR9) and Ly-6B.2 (Novus, NBP2-13077) staining was performed according to the manufacturer’s instructions. TUNEL (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling) staining was performed using the Dead-End kit (Promega, PRG7130) according to the manufacturer’s instructions.
For human lung samples, de-identified autopsy lung samples were provided by the Asian Institute of Gastroenterology as paraffin embedded tissue blocks. Sectioning (4 μm) and immunohistochemistry were performed by HistoWiz with hematoxylin and eosin, the rabbit polyclonal anti–SARS-CoV-2 Nucleocapsid (N) protein antibody (GeneTex; GTX635686), and TUNEL (Promega) staining following standard protocols.

Mice were sacrificed 24 h after the last e-cigarette exposure and were euthanized by administering a mixture of ketamine and xylazine. Lungs were lavaged via tracheal catheterization three times each with 0.6 mL of 0.05% fetal bovine serum in 0.9% NaCl. The combined lavage fluids were centrifuged at 2000 rpm for 10 min at 4 °C. The supernatant was recovered and stored at −80 °C, while the cell pellet was resuspended in 1 mL of 1 × phosphate buffer saline (PBS). Total cell counts were measured by staining cells with acridine orange and propidium iodide (AO/PI) and counted using the Nexcelom Cellometer Auto 2000 cell viability counter. Differential cell counts were determined by flow cytometry using a Guava easyCyte flow cytometer with a minimum of 100,000 cells per sample. Cells from BALF were stained with CD16/32 (Tonbo biosciences 70-0161-u500, 1:10) to block nonspecific binding and then cells were stained using a master mix of CD45.1 (Biolegend Cat# 110728, 1:1000, San Diego, CA, USA), F4/80 (Biolegend Cat# 123110, 1:500, San Diego, CA, USA), Ly6 B.2 (Novus Biological Cat# NBP2-13077, 1:250, Littleton, CO, USA), CD4 (Invitrogen Cat# 25-0041-82, 1:500, Carlsbad, CA, USA), and CD8 (Invitrogen Cat# 17-0081-82, 1:500, Carlsbad, CA, USA).