Cells were preextracted with CSK-T [10 mM Pipes (pH 7), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5% Triton X-100], fixed with 2% formaldehyde, and processed as described (7). EdU staining was performed using the Click-iT EdU Alexa Fluor 488/647 High-Throughput Imaging (HCS) Assay Kit (Invitrogen) according to the manufacturer’s instructions. In brief, cells were labeled with 40 μM EdU for 15 min, preextracted, fixed, and imaged. The treatment of cold methanol at −20°C for 15 min was used for antigen retrieval of endogenous PCNA. Images were collected using a DeltaVision system (Applied Precision) with UApo/340 40×/1.35 numerical aperture (NA) oil objective lens or ScanR system with UPlanSApo 20×/0.75 NA objective lens. All images in the individual panels were acquired under room temperature with the same settings and adjusted for brightness and contrast identically using Adobe Photoshop CS6. Analysis was done with SoftWoRx software (Applied Precision), Volocity image analysis software (PerkinElmer), or ScanR analysis software. All experiments were carried out in biological triplicates, and mean intensities are displayed in graphs unless otherwise indicated.

For HTM, cells grown in Lab-Tek II Chamber slides (Labclinics) were preextracted in 0.2% Triton X-100/phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA). Fixed cells were blocked in 3% bovine serum albumin (BSA)/0.1% Tween/PBS for 1 hour and stained. Forty-eight images per well were automatically acquired with a robotized fluorescence microscopy station (ScanR, Olympus) at ×40 magnification and nonsaturating conditions. Images were segmented using the DAPI staining to generate masks matching cell nuclei from which the corresponding signals were calculated using an in-house–developed package based on CellProfiler. Antibody information and technical details can be found in Supplementary Materials and Methods.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.