Six strains of P. insidiosum from different sources were selected; ATCC 58643 (CBS 574.85) from an equine, CBS 101039 from an Indian patient, CBS 777.85 from an equine, PC10 from a Thai patient, ATCC 90586 from an American patient, and PEC1 from water environment in Pasak Chonlasith Dam, Thailand. These isolates were grown in Sabouraud dextrose broth (SDB, Oxoid, UK). The zoosporogenesis technique was modified from Mendoza and Prendas [22 (link)]. The pathogens were cultured on sterile grass leaves (Axonopus compressus). After incubating at 37˚C for 48 h, grass leaves with hyphae were further transferred into the induction medium to generate encysted zoospores. The induction medium contains calcium and magnesium ions that facilitate the development of zoospores by all Pythium species [5 (link)]. The encysted zoospores were harvested from the induction medium and washed with 1x phosphate-buffered saline (PBS, Biolegend, USA). Then, zoospore numbers were counted by a hemocytometer. All six strains of zoospores were individually used in all experiments; neutrophil killing assay, phagocytosis, and NET formation.
Phosphate buffered saline pbs
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Phosphate-buffered saline (PBS) is a commonly used buffer solution in various laboratory applications. It is a isotonic solution that maintains a physiological pH and ionic concentration, suitable for preserving the natural state of biological samples. PBS is a versatile reagent that supports the stability and viability of cells, proteins, and other biomolecules during various experimental procedures.
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2 protocols using phosphate buffered saline pbs
1
Zoospore-mediated Pathogenicity of Pythium insidiosum
2
Obesity model and IL-25 intervention
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C57BL/6 wild-type mice were purchased from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China). The care, use and treatment of all animals in this study were in strict accordance with the principles set forth by the Animal Care and Use Committees of Sun Yat-sen University. For these experiments, 4- to 5-week-old male mice were fed a normal chow diet (NCD) or a high-fat diet (HFD, 60% of calories, D12492, Research Diets Inc., NJ, USA) for 3 months to create the obesity model, and body weight and food consumption were monitored. At the end of 3 months of the NCD or HFD-induction program, mice were intraperitoneally injected with bovine serum albumin (BSA) or IL-25 (1 μg in 100 μl phosphate-buffered saline (PBS) for each mouse, Biolegend, San Diego, CA, USA) every day for 7 days followed by every other day for an additional 16 days for a total of 23 days. During the HFD treatment, the serum and tissues of mice were also collected at 0, 1, 2, 4, 12 and 16 weeks to explore changes in the level of IL-25. DB/DB (Lepr−/−) mice were purchased from the Nanjing Laboratory Animal Center (Nanjing, China).
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