Prolong antifade mounting agent

MEFs were pretreated with 25μM menadione for 1 hr at 5% O₂ and washed using growth media. Cells were incubated with 1mM 5-bromo-2’-deoxyuridine (BrdU) for 24 hr to allow replication and incorporation of the BrdU label in the DNA. Cells were then washed twice with growth media and the incorporated BrdU label was chased for 0 hr or 24 hr. The 0 hr chase samples were considered a measure of total BrdU label that was incorporated in the cells. Cells were then fixed using 2% formaldehyde for 10 min, treated with 50mM ammonium chloride for 5 min and permeabilized using 0.2% Triton X-100 for 10 min. To allow antibody access to the incorporated BrdU, DNA hydrolysis was performed by incubating cells with 1N HCl for 10 min at 4°C followed by 1N HCl for 10 min at RT. Cells were washed with 0.2% Triton X-100, blocked in 2% BSA for 20 min and incubated with Alexa Fluor 488 conjugated anti-BrdU antibody (Biolegend) for 30 min. Cells were washed with PBS and mounted using DAPI containing Prolong antifade mounting agent (Invitrogen). Cells were imaged using a Deltavision deconvolution microscope. At least 20 fields of view were imaged per sample. Results were compiled from 3 independent experiments.

RPMI-1640, Ammonium-Chloride-Potassium (ACK) lysing buffer, EDTA 0.5M, and HEPES 1M were purchased from Lonza (Walkersville, MD). MEM nonessential amino acids (NEAA), L-glutamine, Penicillin/streptomycin, Hanks’ Balanced Salt Solution with Ca²⁺ and Mg²⁺ (HBSS⁺) or HBSS without Ca²⁺ and Mg²⁺ (HBSS⁻), and Phosphate Buffered Saline with (PBS⁺) or without Ca²⁺ and Mg²⁺ (PBS⁻) were purchased from Corning Cellgro, Mediatech Inc. (Tewksbury, MA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Oakwood, GA). Leukotriene B₄ (LTB₄) was from Cayman Chemical (Ann Arbor, MI). N-Formylmethionyl-leucyl-phenylalanine (fMLF), DNase I, Histopaques-1077 and −1119, 2,2'-azino-di-(3-ethyl)di-thiazoline-sulfonic-acid (ABTS), DuoLink^® In situ-Fluorescence kit, and proteinase inhibitor cocktail (cat#P8340) were from Millipore-Sigma (St. Louis, MO). Recombinant Protein G-Sepharose^® 4B, Hoechst 33342, CountBright counting beads, and Prolong antifade mounting agent were from Invitrogen (Carlsbad, CA). Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN). Recombinant mouse TNF-α and IFN-γ were purchased from Peprotech (Rocky Hill, NJ). A complete list of antibodies used in this work is provided as supplemental material.