Csu x1 spinning disk confocal system

Live-cell imaging of SMP1-1–GFP-expressing T. cruzi was performed as follows.
(i) Amastigotes. Approximately 2 × 10⁶ freshly isolated intracellular T. cruzi amastigotes in 150 μL of prewarmed imaging medium were placed directly on the glass coverslip of a 35-mm glass-bottom dish and allowed to settle at 37°C in a 5% CO₂ incubator for 10 min. Seventy-five microliters of medium was removed prior to placing the dish on the microscope for imaging.
(ii) Epimastigotes. Approximately 2 × 10⁵ epimastigotes in 150 μL of LIT growth medium were placed directly on the glass coverslip of a 35-mm glass-bottom dish and allowed to settle prior to imaging. Parasites were imaged using a Yokogawa CSU-X1 spinning disk confocal system paired with a Nikon Ti-E inverted microscope and an iXon Ultra 888 EMCCD camera (100× objective). Image processing, analysis, and display were performed using ImageJ Fiji software (55 (link)).

Trypanosoma cruzi epimastigotes expressing CYP51::GFP or ∆CYP51 epimastigotes were fixed in 1% paraformaldehyde and permeabilized with TritonX-100. Parasites were placed on poly-L-lysine coated slides and stained with a rabbit α-BiP primary antibody (gift from Jay Bangs, 1:1,000 dilution; Bangs et al., 1993 (link)), and a goat α-rabbit secondary antibody conjugated to Alexa Fluor 647 (Invitrogen, Waltham, MA, United States of America 1:1,000 dilution). DAPI (Thermo Scientific, Waltham, MA, United States of America (1:5,000 dilution, 1 mg/ml stock) was used to identify parasite DNA. Parasites were mounted in ProLong Diamond (Thermo Fisher, Waltham, MA, United States of America) and cured for 24 h. Parasites were imaged on a Yokogawa CSU-X1 spinning disk confocal system paired with a Nikon Ti-E inverted microscope and an iXon Ultra 888 EMCCD camera. The 100x lens was used for imaging, and image processing, analysis, and display were completed in FIJI (Schindelin et al., 2012 (link)).