Growth was assessed by determining plaquing efficiency and growth rate (LaFavers et al., 2017 (link)). For doubling assays, 100,000 parasites were used to infect confluent HFF monolayers grown in 12-well tissue culture plates. Two-hours after infection, cultures were washed three times to remove parasites that did not attach or invade and the media was replaced with normal growth medium. At either 24 or 48 hours post infection, cells were fixed with methanol for 1 minute, dried, and stained using Differential Quik Stain kit (Polysciences, Inc). Number of parasites per vacuole was scored for at least 100 vacuoles for each well. For plaque assays, 500 parasites were used to infect a confluent HFF monolayer in 12-well plates. Infected cultures were incubated for 5 days and stained with Crystal Violet (Sigma Aldrich) to visualize plaques via imaging with a FluorChem R imager (Bio-Techne).
Fluorchem r imager
Manufactured by Bio-Techne
The FluorChem R imager is a laboratory instrument designed for imaging and analysis of fluorescent and chemiluminescent samples. It offers high-resolution imaging capabilities and supports a variety of fluorescent and chemiluminescent detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (1 mentions)
Differential quik stain kit, by Polysciences (1 mentions)
Trans blot sd semi dry transfer system, by Bio-Rad (1 mentions)
4 to 20 mini protean tgx gels, by Bio-Rad (1 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
2 protocols using fluorchem r imager
1
Measuring Toxoplasma gondii Plaquing and Growth
2
Western Blot Analysis of Parasite Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly egressed parasites were passed through 3.0-μm filters to remove host cell debris and then washed in phosphate-buffered saline (PBS). Parasites were lysed in radioimmunoprecipitation assay buffer supplemented with complete protease inhibitor cocktail (Roche), sonicated twice for 10 s each time with a microtip sonicator, and centrifuged to remove insoluble debris. Cleared lysate was subjected to SDS-PAGE with precast 4 to 20% Mini-PROTEAN TGX gels (Bio-Rad), and resolved proteins were transferred to nitrocellulose membranes with the Transblot SD semidry transfer system (Bio-Rad). Membranes were blocked in 5% milk–Tris-buffered saline–Tween 20 (TBST) and probed with primary antibodies for 1.5 h at room temperature or overnight at 4°C. Membranes were washed three times for 10 min each time in TBST and probed with secondary antibodies for 45 min at room temperature. Membranes were washed again three times for 10 min each time in TBST, and proteins were detected with SuperSignal West Femto substrate (Thermo Fisher) and imaged on a FluorChem R imager (Bio-Techne).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!