
Explore the MTT assay, a colorimetric test using tetrazolium salt to measure viable cells and cytotoxic effects, detailing the three-day protocol and analysis of formazan absorbance.
Plan day one of MTT assay on a 96-well plate by outlining compounds, dilutions, replicates, and controls. Use a hemocytometer and trypan blue to estimate cells and calculate plating volumes.
On day two of the MTT assay, prepare stock solutions and dissolve samples. Use C1V1=C2V2 to dilute, plate 100 μl in 96 wells, and incubate at 37°C with 5% CO2.
Prepare MTT dye in PBS on day 3, add 15 µl per well in a 96-well plate, incubate 2.5–4 hours, dissolve Formazan with DMSO, and read at 550 nm.
Sort plate reader data from a 96-well plate by compound and dilution; calculate percentage viability and cytotoxicity using blanks and controls, then create Excel graphs showing dose-dependent cytotoxicity with replicates.
Explore the wound healing, scratch, or cell migration assay as an in vitro method to measure cell movement in a monolayer and its applications in cancer, chemotaxis, and drug screening.
Explore the antiviral assay in mammalian cell culture, evaluating compounds for their ability to prevent cytopathic effects and assess toxicity across dilutions to identify viral targets and aid drug development.
Carry out step one of antiviral assay by inducing cytopathic effect in cell lines (eg, Vero) with dengue, and monitor virus-induced changes daily to prepare propagated virus.
Assess antiviral assay results by comparing treated and control cells for cytopathic effect and antiviral activity, using real-time PCR, plaque assays, or MTS to gauge dengue titer.
This course is designed for undergraduate and graduate biotechnology students who possess an inclination towards acquiring knowledge in basic cell culture techniques but lack a starting point. This resource will primarily assist students with some basic background in mammalian cell culture. You will acquire comprehensive knowledge of different basic but important cell culture assays and techniques. This course is designed for individuals who have a background in biotechnology, immunology, genetics, proteomics, cancer biology, microbiology, molecular biology, stem cell biology, cell and gene therapy, and animal cell culture.
The purpose of this course is to provide an overview of the assays of mammalian cell culture. The initial lessons of the program provide a fundamental introduction to MTT assay (a critical technique in cell culture), including introduction, three-day protocol, and results analysis, as well as the significance and applications in the field of biotechnology. The second section introduces the wound healing or scratch assay, along with its detailed methodology and results analysis. The third section covers the introduction, importance, methodology, and analysis of Antiviral assay.
Although there are certain similarities in the fundamentals of cell culture experiments, the conditions under which cells are cultured differ significantly between labs and cell types. Altering the culture conditions specified for a given cell line may lead to the expression of distinct phenotypes. Consequently, it is advisable that researchers acquaint themselves with the cell line of interest and diligently adhere to the instructions included with every product utilized in their experiments.
(Note: The protocols provided in the course are performed by the instructor but can be changed according to the lab conditions.)
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