Ultra 888 emccd camera

For the Ca²⁺ flux experiments, T cells (~10⁶) were incubated with 5 μM of the fluorescent dye Fluo-4 AM (Thermo Fisher Scientific) for 30 min in complete RPMI 1640 medium. All the Fluo-4 loading and imaging experiments were performed in the presence of 2.5 mM probenecid. The T cells were washed twice with minimal imaging media (MIM; colorless RPMI with 5% FBS and 10 mM HEPES) and then incubated in MIM for 10 min at 37 °C before data collection.^{58 (link)} For imaging, a LEITZ DMIRB Leica Microscope equipped with a 100× objective and an iXON Ultra 888 EMCCD camera were used. The calcium flux imaging acquisition was performed with Micromanager software. For the T cell/APC conjugate experiments, CH27 cells (10⁶) were incubated with 4 μM of each peptide for 4 h at 37 °C and then washed with MIM. The T cells (2 μL) and CH27 cells (2 μL) were added to MIM (300 μL) in the cell chamber. The chamber was sealed using mineral oil on both sides to avoid MIM evaporation. The signals from Fluo-4 were collected at intervals of 100 ms for up to 20 min and postprocessed with Fiji software.

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)).

Cell migration was stimulated by making an infinite scratch wound. The cells were allowed to recover for a period of 2 hr before imaging. Migration rates were measured as the area covered by the edge of the wound in the field of view per unit time using Fiji (NIH, Bethesda, MD). For measurements of directionality in single cell migration assays, primary MEFs were cultured on glass bottom MatTek (Ashland, MA) dishes coated with 5 µg/ml Fibronectin at low cell densities. 2 hr after seeding, cells were imaged at 10 min intervals for 4 hr. Single cells were tracked using Metamorph Track Objects (Molecular Devices, Sunnyvale, CA) module. The obtained total displacement was divided by total distance of the track to obtain a directionality score shown in Figure 5. All images for these experiments were acquired on a Nikon Ti microscope with a 10X Phase objective and Andor iXon Ultra 888 EMCCD camera.

Parasites were fixed in 1% PFA-PBS for 10 min, spotted onto poly-L-lysine coated slides and allowed to adhere for 30 min. Adhered parasites were washed with PBS and permeabilized with 0.1% Triton X-100-PBS for 10 min, rewashed again with PBS, and blocked with 3% BSA (Sigma-Aldrich) in PBS for 1 h. Then, parasites were incubated with anti-BIP antibodies in 1% BSA in PBS for 1 h (a generous gift from Dr J. D. Bangs, U. Buffalo) and washed three times with PBS before incubation for 1 h with goat anti-rabbit IgG secondary antibody Alexa fluor 594 (Thermo Fisher Scientific), and 100 ng/ml DAPI (Sigma-Aldrich). Finally, parasites were washed with PBS and mounted with ProLong Antifade (Thermo Fisher Scientific). EPIs were analyzed using a Yokogawa CSU-X1 spinning disk confocal system paired with a Nikon Ti-E inverted microscope equipped with an iXon Ultra 888 EMCCD camera. All images were acquired with the 100× objective and image processing was completed using ImageJ Fiji (https://fiji.sc) (57 (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)).

Images were acquired on a Nikon TIRF microscope at a TIRF angle of 61° to achieve HILO illumination. Samples were recorded with an iXon Ultra 888 EMCCD camera, filter cube TRF49909-ET-561 laser bandpass filter and 100× oil 1.49 NA TIRF objective. Cells were imaged using a 561nm excitation laser at a power density of 10.3 μW to perform two different acquisition techniques. A fast frame rate which uses a 50 Hz (20 ms acquisition speed) to acquire 6000 frames without intervals to measure displacement distribution and fraction bound, and a slow frame rate which uses a 2 Hz (500 ms acquisition speed) to acquire 500 frames without intervals to measure residence times.