Cholesterol Trafficking Quantification

Cells were seeded on 4-chamber glass slides and grown in 1% FBS DMEM overnight. Treatment with CQ was carried out for 16 hours followed by addition of 1μg/ml CholEsteryl-BODIPY dissolved in DMSO for 4 hours. Cells were fixed using 4% PFA. Immunofluorescent labeling was performed using Lamp2A and Alexa-488 antibodies (Invitrogen, Carlsbad, CA, USA). To quantify CholEsteryl-BODIPY an outline was drawn around each cell (n≥15) and circularity, area and mean fluorescence were measured, along with several adjacent background readings. Total corrected cell fluorescence (TCCF) = integrated density - (area of selected cell × mean fluorescence of background readings) [46 (link)]. The colocalization signal for each condition was measured from four representative fields, n≥15 cells, using ImageJ. The colocalization index represents the Pearson's coefficient (zero is no colocalization and one means perfect colocalization).