Orca r2 digital ccd camera c10600

Tissue culture glass slides with 8 chambers were treated with 0.1% Poly-Lysine (Sigma, P-8920). 2.5 × 10⁶ formaldehyde-fixed T. brucei were allowed to adhere to Poly-Lysine-treated chambered slides (Falcon, 354108), permeabilised with 0.2% (w/v) Triton X-100 then incubated with protein-specific antibodies followed by fluorescently-labelled second antibodies in PBS containing 0.5% gelatin. DNA was stained with 100 ng/ml DAPI (D9542, Sigma-Aldrich). Mitochondria were detected by addition of Mitotracker Red CMXRos (50 nM, Thermo Fisher Scientific) to the cells 5 min prior to fixation. Images were examined using the Olympus IX81 microscope, 100x Oil objective with a numerical aperture of 1.45. Digital images were taken with ORCA-R2 digital CCD camera C10600 (Hamamatsu) and using the Xcellence RT software. The bright-field images were taken using differential interference contrast (DIC). Fluorescent images were taken as Z-Stacks with a height of roughly 4 µm and a step width of 0.2 µm. The images were deconvoluted (Wiener Filter, Sub-Volume overlap: 20) and then processed using ImageJ. The background was subtracted and brightness and contrast were adjusted automatically. The most in-focus image of the deconvoluted stack was used.

Cells were seeded at 1.5 × 10⁵ per 6‐well plate on 13 mm coverslips and the next day treated or not with CldU (1 or 10 μM) or IdU (10 μM). Cells were washed with PBS, fixed in 4% formaldehyde for 15 min, followed by two washes in PBS and denaturation (2 M HCl, 0.5% Triton X‐100) for 30 min. Next, cells were neutralized in 0.1 M Na₂B₄O₇ for 5 min, followed by two washes in PBS. Cells were blocked in BSA in PBS for 1 h followed by incubation with primary rat anti‐BrdU (CldU; Abcam, ab6326, 1:200) or mouse anti‐BrdU (IdU; Becton Dickinson, 347580, 1:200) for 2 h and extensive washing. Samples were incubated in secondary goat anti‐rat Alexa Fluor 568 (Thermo Fisher, A11077; 1:1,000) or goat anti‐mouse Alexa Fluor 488 (Thermo Fisher, A32723; 1:1,000) for 1 h, followed by extensive washing. DNA was counterstained with DAPI and the coverslips were mounted using Mowiol (Sigma Aldrich) mounting media. Images were acquired using an automated Olympus ScanR system, a motorized Olympus IX81 microscope with 40×/0.6 (LUCPLFLN 40× PH) dry objectives and Hamamatsu ORCA‐R2 digital CCD camera C10600. Images were collected and analyzed using ScanR automated software to quantify the fluorescence intensity of individual cell nuclei. All quantification was conducted on cells/samples (typically > 1,000 cells per sample per experiment) selected by scanR software in a random/unbiased manner.