Dmr wide field epifluorescence microscope

Whole-cell FACS was carried out as described in detail in Sabatinos and Forsburg (2009 , 2015 ) and Sabatinos et al. (2015 (link)) with minor changes. To perform cell cycle analysis or microscopy, cells were fixed in 70% ethanol. Following rehydration in 50 mM sodium citrate, cells were treated with 1 µM SytoxGreen (Invitrogen, Carlsbad, CA) plus 10 µg/ml RNase A and incubated at 36°C for 1–2 h. Samples were then sonicated before being analyzed on a FACScan machine (BD Biosciences, San Jose, CA). DAPI staining was done by first rehydrating fixed cells in PBS and placing them to dry on positively charged glass slides. Mount solution (50% glycerol and 1 µg/ml DAPI) was then applied before cells were photographed on a Leica DMR wide-field epifluorescence microscope equipped with a 63×/1.62 NA Plan-Apo objective lens, 100-W Hg arc lamp for excitation, and a 12-bit Hamamatsu ORCA-100 CCD camera. Images were acquired using OpenLab version 3.1.7 (ImproVision, Lexington, MA) software and analyzed with ImageJ.

Cells were fixed in cold 70% ethanol for cell cycle analysis or microscopy. For DAPI/septa staining, cells were rehydrated in water and incubated for 10 min in 1 mg/ml aniline blue (M6900; Sigma-Aldrich). Cells in mount (50% glycerol, 1 μg/ml DAPI, and 1 μg/ml p-phenylenediamine) were photographed on a Leica DMR wide-field epifluorescence microscope using a 63× objective lens (numerical aperture [NA] 1.62 Plan Apo), 100-W Hg arc lamp for excitation, and a 12-bit Hamamatsu ORCA-100 charge-coupled device (CCD) camera. OpenLab version 3.1.7 (ImproVision, Lexington, MA) software was used at acquisition and ImageJ (National Institutes of Health, Bethesda, MD) for analysis.
Whole-cell SYTOX Green and EdU flow cytometry (fluorescence-activated cell sorting [FACS]) were performed as described in Sabatinos et al. (2012 (link), 2013 (link)). DNA synthesis by EdU incorporation was assessed by adding 10 μM EdU to cultures before harvest. Whole-cell FACS for EdU was performed on rehydrated cells using Click-iT (Invitrogen, ThermoFisher Scientific) with Alexa Fluor 488.