L alanine l glutamine

Human normal monocytes (CD14⁺ cells) were isolated by the AutoMACS Pro Separation System (Miltenyi Biotech, Auburn, CA) from the mononuclear cells fraction of normal peripheral blood. Briefly, buffy coat was purchased from Interstate Blood Bank (Memphis, TN) and the mononuclear cell layer was separated by Ficoll Hypaque (17144003, GE Lifesciences) density gradient separation. Mononuclear cells were treated with red blood cell lysis buffer to remove red blood cells and then incubated with CD14 microbead-conjugated antibody (130–050-201, Miltenyi Biotech, Auburn, CA) for 15 min at 4°C. CD14 positive cells were then isolated using the positive selection program according to the manufacturer’s protocol. One million CD14⁺ cells were plated in macrophage culture media, Iscove’s modified Dulbecco’s medium (IMDM) (12440053, Thermo fisher) supplemented with 10% human AB serum (MT35060CI, Fisher Scientific), 1% NEAA (11–140-050, Fisher), 2 μM L-alanine-L-glutamine (SH3003402, Fisher), per each well of a 6-well plate and cultured for 7 days. After incubation, most of the cells were adherent to the plastic surface and stained positive for CD14 and other markers specific for macrophages.