Rolera em c2 camera

BAC100, alone or in combination with antibiotic-treated MRSA cells, was analysed for live and dead cells together with the control according to the protocol described by Manoharadas et al. [28 (link)]. Briefly, SYTO9: Propidium iodide (Thermo Fisher Scientific, Waltham, MA, USA) dye at a dilution of 1:1000 (5 μM: 5 nM) was added to the MRSA cells. The mixture was incubated for 10 min at room temperature under dark conditions. The mixture was centrifuged for 2 min and the supernatant was discarded. The pellet was washed thrice with 1× PBS buffer. The stained cells were imaged using a spinning disk confocal microscope (Zeiss, Jena, Germany) after mounting onto a glass slide covered with a cover slip. Live cells were imaged with SYTO9 dye (green cells) at excitation/emission wavelengths of 483/503 nm. Similarly, dead cells were imaged with propidium iodide (red cells) at excitation/emission wavelengths of 535/617 nm. A Rolera Em-C² camera (Zeiss, Jena, Germany) with an oil immersion objective lens specification of 63× was used for image acquisition. The captured images were processed using the Zen lite software (ZEN 3.1 (blue edition), Zeiss, Jena, Germany). The numbers of live (green) and dead (red) cells per frame/picture were counted manually. The experiment was performed in triplicate and the mean value was calculated.

The live–dead staining of bacterial cells was performed according to the protocol described by Manoharadas et al., 2021 [31 (link)]. Briefly, the staining for viable cells was performed by the addition of 5 μM of SYTO9 (ThermoFischer Scientific, Bedford, MA, USA), diluted in DMSO. The staining was performed for 10 m in dark conditions. After incubation, the cover slips were further washed extensively with 1× PBS and final rinsing in double distilled water. In order to stain for nonviable cells, propidium iodide (ThermoFischer Scientific, Bedford, MA, USA) was diluted in 2× SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) to a final concentration of 500 nm. The diluted propidium iodide was added to the cells. The staining was performed for 10 m, followed by washing with 1× PBS and final rinsing with double distilled water. The image was captured by confocal microscope (Zeiss, Jena, Germany) at an excitation/emission of 483/503 nm for SYTO9 and 535/617 nm for propidium iodide. The images were acquired on a Rolera Em-C2 camera with a 63× oil immersion objective (Zeiss, Jena, Germany). The acquired image was processed by the Zen lite software (Zeiss, Jena, Germany).

LLC-MK2 cells were infected with cell-culture derived Y-strain trypomastigotes at 10:1 ratio overnight at 37°C, 5% CO₂. After 2 days, cells were fixed in 4% paraformaldehyde/PBS for 15 min and permeabilized with three washes with PBS-0,1% Triton X-100 (Bio-Rad). Sera of immunized and/or infected mice, as well as of naïve animals were added and incubated overnight at 4°C. After washing three times with PBS-0, 1% Triton X-100, donkey Cy3-labeled anti-mouse IgG was added for 2 h at RT. After washing, DAPI was added at 1 µg/ml at RT for 5 min. The coverslips were assembled with vectashield and fixed with enamel. Confocal microscopy was performed with a Zeiss Axio Observer.Z1 inverted microscope equipped with a CSU-X1A 5000 Yokogawa Spinning Disk confocal unit using a 100 × NA 1.4, oil-immersion plan-apochromatic objective. Images were captured with QImaging Rolera EM-C2 camera using Zen 2.3 system (Zeiss), and processed off line with Photoshop.