Acridine orange propidium iodide

Unless specified as counted on-plate, cells were counted using a Cellometer K2 (Nexcelom) after lifting from culture dishes. H1299 cells and primary human fibroblasts were counted using a 1:4 dilution of trypan blue (Thermo Fisher), while BMDMs were counted using a 1:1 dilution of acridine orange/propidium iodide (Thermo Fisher, VWR). When counting on-plate, 1.25 μM Calcein-AM (final concentration; Santa Cruz Biotechnologies) was added to each well and incubated for 15 minutes. The plates were then imaged on a Molecular Devices ImageXpress XL. 24 high magnification fluorescence images were captured for each well (11.9% of total well surface area) using a 10x objective (Nikon Plan Fluor, 0.3 NA). Cell number was assessed using MetaXpress Software with Powercore using the Multi-wavelength cell scoring module. For collection with guanidine HCl, cells were treated with 50 μL or 75 μL of 6 M aqueous guanidine HCl and transferred to glass tubes for derivatization with an additional 100 μL or 150 μL of 3 M methanolic guanidine HCl (24-well plates and 12-well plates, respectively). Samples were prepared alongside internal standard curve samples made up of FAMEs (Nu-Chek Prep).