C. parvum sporozoites were tested for viability by modifying a previously described assay [24 (link)]. Briefly, 5 × 106 oocysts were bleached, washed with 1X PBS and the oocysts suspended in 0.6% taurocholate acid in cell culture medium. Oocysts were excysted at 37 °C for 30 min with mixing every 5 min. Excysted oocysts and sporozoites were aliquoted and treated with either 100 nM trtE or DMSO for 1 h at 37 °C. For a positive control for killing, sporozoites were either treated with 10% formaldehyde and heat shocked for 2 min at 60 °C or heat shocked only. To label viable parasites, treated sporozoites were then incubated with CFSE a final concentration of 10 µM (Invitrogen, C1157) at 37 °C for 30 min. DNA was labeled with 0.9 mM Hoechst (Abcam, ab1455971). Samples were evaluated by fluorescence microscopy (Zeiss® Axioscope 5). Slides were blinded and the number of viable sporozoites per 200 sporozoites were counted for each sample. The Jenoptik GRYPHAX® camera and its image acquisition software (v2.2.0) were used to take representative images of sporozoites.
Gryphax camera
Manufactured by Jenoptik
The GRYPHAX® camera is a high-performance digital camera designed for laboratory applications. It features a CMOS sensor and provides high-resolution image capture capabilities. The camera is compatible with a variety of microscopes and offers connectivity options for easy integration into laboratory workflows.
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Lab products found in correlation
4 protocols using gryphax camera
1
Viability Assay for C. parvum Sporozoites
2
Histological Evaluation of Muscle Biopsy
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Histological evaluation of muscle biopsy for a hard‐to‐treat patient (referred to as Patient 3, see Table 1 ) was performed by conventional light microscopy. Specimens were processed according to established clinical diagnostic histopathological procedures performed by CLIA (Clinical Laboratory Improvement Amendments)‐accredited Pathology Laboratory of UPMC Clinical Laboratory Services. Frozen sections of biopsy samples were subjected to routine H&E and immunoperoxidase techniques employing antibodies specific for tissue macrophages (CD68, clone PGM1, Roche), endothelial cells (CD31, clone JC70A, Dako Agilent) and MHC I (anti‐HLA‐A, clone EP1395Y, Abcam). Immunostaining included isotype controls. PAS staining was also carried out. All immunohistochemically stained slides were counterstained with haematoxylin. Slide preparations utilised the Benchmark ULTRA System (Roche Diagnostics). Histological evaluation was performed by a paediatric pathologist (author MRM) using a bright‐field Olympus® BX51 microscope, and images were obtained with a Jenoptik® Gryphax camera. Raw images were used without manipulation.
3
Visualizing Osx-Cherry Embryo Expression
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Embryos were dissected in phosphate-buffered saline and visualized using a Leica MZ10F fluorescent dissecting microscope for Cherry fluorescence. Images were captured using a JENOPTIK GRYPHAX camera. Frozen sections of Osx-Cherry embryos were visualized for Cherry and DAPI fluorescence using an A1 Nikon confocal microscope.
4
Inhibition of C. parvum Invasion in HCT-8 Cells
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This method was modified from an assay described previously [11 (link)]. Briefly, HCT-8 cells were seeded on cell culture chamber glass slides (NEST® Scientific, Woodbridge, NJ, USA) until confluent and treated for 1 h with either trtE, DMSO, or wiskostatin. Wiskostatin was reported to inhibit C. parvum invasion [11 (link)] and is used here as a control. HCT-8 monolayers were then infected with 50,000 NLuc oocysts per well. Heat inactivated oocysts were used as negative control. After 3 h of incubation, cells were washed three times with 111 mM D-galactose in 1X PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.25% Triton-100X. Staining of parasitophorous vacuoles was performed with 1.33 µg/mL of fluorescein-label Vicia villosa lectin (VVL) (Vector Laboratories, Newark, CA, USA). Nuclei staining was performed with 0.09 mM Hoechst. Slides were blinded and stained vacuoles from at least 10 randomly selected fields were counted in each sample at 40X magnification (Zeiss® Axioscope 5). Percent inhibition was normalized to DMSO as described above. The Jenoptik GRYPHAX® camera and its image acquisition software (v2.2.0) were used to take representative images of infected monolayers.
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