Uplsapo 60.0x 1 35 oil objective

In brief, HeLa cells were grown on a coverslip in a 6 well plate 24 hr prior to treatment. HeLa cells were treated with indicated siRNAs for 48 hr. For GFP-RNase H1 overexpression, plasmid transfection were performed at 24 hr post siRNA treatment, followed by incubation for a further 24 hr. HeLa cells were fixed with 4% PFA in PBS. Primary antibody anti-γH2A.X (JBW-301) was used at 1:200 in 3% BSA in PBS for 1 hr at room temperature. Cells were washed thrice with 0.05% Tween20-PBS followed by incubation with secondary donkey anti-mouse IgG (H+L) conjugated with Alexa Fluor 488 at (1:250) concentration. Z stack images were collected with a FluoView1000 confocal microscope (Olympus) using a UPLSAPO 60.0X / 1.35 oil objective. Images were analyzed using ImageJ and prepared using OMERO software. For γH2A.X foci quantification, approximately 10 unique fields of view from distinct images, were captured at random. Binary images were thresholded and water shed once with area of each foci was determined using the ‘Analyze Particles’ feature of ImageJ. Cut-off of particle size was set to (> 200 nm2-infinity) and circularity (0.5-1.10). The percentage of foci > 200nm2 were scored as positive.