Chlamydia was propagated and purified using mouse fibroblast McCoy cells (McCoy B, CRL-1696™) as described previously (34 (link), 35 (link)). Briefly, supernatant-derived Chlamydia was purified by ultracentrifugation of supernatants harvested from the infected McCoy cells at 22,500×g for 1 h at 4°C. Monolayer-derived Chlamydia was purified following the lysis of McCoy monolayers by sonication and density gradient separation with 30% of the Isovue solution (ISOVUE® iopamidol injection) and 50% sucrose dissolved in 30 mM Tris HCl at 22,500×g for 1 h at 4°C. Purified Chlamydia was quantified by an IFU assay. McCoy cells infected with serially diluted standards or samples were fixed in 100% methanol and stained with Gimenez solution (1 g of carbol fuchsin dissolved in 10 ml of ethanol, 4% phenol dissolved in distilled water) at room temperature for 20 s. The cells were then counterstained with 0.8% Malachite Green solution, and IFU was assessed visually. Chlamydia trachomatis serovar E (ATCC VR 348B™) was used for human mast cell experiments and Chlamydia muridarum (ATCC VR-123™) was used for murine mast cell experiments and in vivo infection experiments.
Vr 348b
Manufactured by American Type Culture Collection
The VR 348B is a laboratory centrifuge designed for a variety of applications. It is capable of maintaining consistent speeds and temperatures to ensure accurate and reliable results during the centrifugation process. The core function of this equipment is to separate and isolate cellular components, biomolecules, or other materials based on their density differences when subjected to centrifugal force.
Automatically generated - may contain errors
2 protocols using vr 348b
1
Chlamydia Propagation and Purification
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Chlamydia was propagated and purified using mouse fibroblast McCoy cells (McCoy B, CRL-1696™) as described previously (34 (link), 35 (link)). Briefly, supernatant-derived Chlamydia was purified by ultracentrifugation of supernatants harvested from the infected McCoy cells at 22,500×g for 1 h at 4°C. Monolayer-derived Chlamydia was purified following the lysis of McCoy monolayers by sonication and density gradient separation with 30% of the Isovue solution (ISOVUE® iopamidol injection) and 50% sucrose dissolved in 30 mM Tris HCl at 22,500×g for 1 h at 4°C. Purified Chlamydia was quantified by an IFU assay. McCoy cells infected with serially diluted standards or samples were fixed in 100% methanol and stained with Gimenez solution (1 g of carbol fuchsin dissolved in 10 ml of ethanol, 4% phenol dissolved in distilled water) at room temperature for 20 s. The cells were then counterstained with 0.8% Malachite Green solution, and IFU was assessed visually. Chlamydia trachomatis serovar E (ATCC VR 348B™) was used for human mast cell experiments and Chlamydia muridarum (ATCC VR-123™) was used for murine mast cell experiments and in vivo infection experiments.
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Chlamydia was propagated and purified using mouse fibroblast McCoy cells (McCoy B, CRL-1696™) as described previously (34 (link), 35 (link)). Briefly, supernatant-derived Chlamydia was purified by ultracentrifugation of supernatants harvested from the infected McCoy cells at 22,500×g for 1 h at 4°C. Monolayer-derived Chlamydia was purified following the lysis of McCoy monolayers by sonication and density gradient separation with 30% of the Isovue solution (ISOVUE® iopamidol injection) and 50% sucrose dissolved in 30 mM Tris HCl at 22,500×g for 1 h at 4°C. Purified Chlamydia was quantified by an IFU assay. McCoy cells infected with serially diluted standards or samples were fixed in 100% methanol and stained with Gimenez solution (1 g of carbol fuchsin dissolved in 10 ml of ethanol, 4% phenol dissolved in distilled water) at room temperature for 20 s. The cells were then counterstained with 0.8% Malachite Green solution, and IFU was assessed visually. Chlamydia trachomatis serovar E (ATCC VR 348B™) was used for human mast cell experiments and Chlamydia muridarum (ATCC VR-123™) was used for murine mast cell experiments and in vivo infection experiments.
2
Propagation and Quantification of Chlamydia trachomatis Serovars
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The C.t. serovar D (C.t. SvD) (UW-3/Cx, ATCC VR-885), SvE (BOUR, VR-348B), and SvF (IC-Cal-3, ATCC VR-346) were purchased from the ATCC and propagated in HeLa-229 cells. Six-well plates were centrifuged at 750 g for 1 h at RT. C.t. elementary bodies (EBs) were harvested, purified, and quantified as described previously (21 (link)) and stored at −80°C in a sucrose-phosphate-glutamate (SPG) buffer. All procedures were done using Biosafety level 2 containments.
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