Quantifying Glucocorticoid-Induced CLAN Formation

MTM cells from GR^dim and WT mice were cultured on glass coverslips in 24-well plates until confluent. Once confluent, they were treated with vehicle control (0.1% ethanol) or DEX (100 nM) for 7 days. At the end of the experiment, cells were processed as described in Immunocytochemistry until the blocking step. F-actin was stained with phalloidin conjugated with Alexa-488 (1:300; Life Technologies, Eugene, OR, USA) at 4°C overnight. After PBS washes, coverslips were mounted on ProLong gold anti-fade reagent with DAPI (Invitrogen-Molecular Probes). CLANs were visualized by using Nikon Eclipse Ti-U microscope with a Nuance imaging system (Nikon, Melville, NY, USA). CLANs were defined as F-actin–containing cytoskeletal structures with at least one triangular actin arrangement consisting of actin spokes and at least three identifiable hubs.72 (link) Each coverslip was assessed at nine locations with approximately 80 to 170 cells evaluated per coverslip. Two to three coverslips were evaluated per treatment group. CLAN-positive cells (CPCs), defined as any cell containing at least one CLAN or multiple CLANs, were calculated as the percentage of CPCs by dividing the number of CPCs by the number of DAPI-positive cells.