Ficoll gradient

C57BL/6 mice (Jackson Laboratories, Bar Harbor, Maine) were anesthetized and then exsanguinated by clipping the axillary/brachial artery. Animals were then perfused with 10 ml of PBS to remove remaining blood. Cells were harvested by first detaching heads and removing skin, lower jaws, palates, muscles, cheek bones and incisors. Remaining snouts including both respiratory and olfactory regions were broken into pieces using a syringe plunger. They were then digested with collagenase type II (Worthington Biochemical Corp, Cat# LS004177, 200 U/ml), dispase (Becton Dickinson and Company, Cat# 354235, 50 U/ml) and DNase (Sigma-Aldrich, Cat# D4513,15 U/ml) for 1 hour at 37°C in a shaker at 225 rpm. Cells were passed through a 100 µm cell strainer and washed 2× with complete medium (CM), a Modified Eagles Medium (Invitrogen, Grand Island, NY) supplemented with dextrose (500 µg/ml), glutamine (2 mM), 2-mercaptoethanol (3×10⁻⁵ M), essential and non-essential amino acids, sodium pyruvate, sodium bicarbonate and antibiotics [21] (link), plus 10% heat inactivated fetal bovine serum (FBS). Dead cells were removed from cell populations with a Ficoll gradient (MP Biomedicals, LLC, Cat# 50494) and Dead Cell Removal beads (Miltenyi Biotech, Cat# 130-090-101). URT cells isolated from nasal tissue by collagenase, dispase and DNase digestions are termed ‘NT’ in this report.