Exercise 3 The MTT assay is a widely used assay to study cell viability. It measures cellular metabolic activity, which serves an indicator of cell viability, proliferation and cytotoxicity. This non- radioactive colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. Viable cells contain NAD(P)H-dependent oxidoreductase enzymes that reduce the MTT to formazan. The insoluble formazan crystals are dissolved and the resulting coloured solution is quantified by measuring absorbance at 500-600nm using a multi-well spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells! Exercise 3.1 MTT protocol Your colleagues in the School of Biology and Environmental Science discovered an unusual looking green algae on a recent fieldtrip to Costa Rica. They have provided you with some extracts from the plant and you're excited to measure their effects on the growth of cancer cells in vitro. You decide to perform an MTT assay on two prostate cell lines: PC3 (metastatic prostate cancer) and PWR1E (immortalised non-cancerous epithelial cells). But "Oh sugar!" your dog ate your lab book! Now you have to try and piece together the correct experimental protocol, which has become all jumbled up. Jumbled up protocol: Incubate plate for 3h. Resuspend cell pellet in cell culture media. Add MTT reagent to each well. Dissolve formazan product using DMSO. Count cells using haemocytometer. Prepare desired concentration of compound for treatment. Seed appropriate number of cells in each well of 96-well plate. Measure absorbance using plate reader. Add treatment to each well of plate and incubate for 72h. Remove media and MTT reagent. Correct experimental MTT protocol: What would be the most appropriate controls to include in this experiment and why? II
Exercise 3 The MTT assay is a widely used assay to study cell viability. It measures cellular metabolic activity, which serves an indicator of cell viability, proliferation and cytotoxicity. This non- radioactive colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. Viable cells contain NAD(P)H-dependent oxidoreductase enzymes that reduce the MTT to formazan. The insoluble formazan crystals are dissolved and the resulting coloured solution is quantified by measuring absorbance at 500-600nm using a multi-well spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells! Exercise 3.1 MTT protocol Your colleagues in the School of Biology and Environmental Science discovered an unusual looking green algae on a recent fieldtrip to Costa Rica. They have provided you with some extracts from the plant and you're excited to measure their effects on the growth of cancer cells in vitro. You decide to perform an MTT assay on two prostate cell lines: PC3 (metastatic prostate cancer) and PWR1E (immortalised non-cancerous epithelial cells). But "Oh sugar!" your dog ate your lab book! Now you have to try and piece together the correct experimental protocol, which has become all jumbled up. Jumbled up protocol: Incubate plate for 3h. Resuspend cell pellet in cell culture media. Add MTT reagent to each well. Dissolve formazan product using DMSO. Count cells using haemocytometer. Prepare desired concentration of compound for treatment. Seed appropriate number of cells in each well of 96-well plate. Measure absorbance using plate reader. Add treatment to each well of plate and incubate for 72h. Remove media and MTT reagent. Correct experimental MTT protocol: What would be the most appropriate controls to include in this experiment and why? II
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
Section: Chapter Questions
Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
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Nucleic Acids
Nucleic acids are essential biomolecules present in prokaryotic and eukaryotic cells and viruses. They carry the genetic information for the synthesis of proteins and cellular replication. The nucleic acids are of two types: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The structure of all proteins and ultimately every biomolecule and cellular component is a product of information encoded in the sequence of nucleic acids. Parts of a DNA molecule containing the information needed to synthesize a protein or an RNA are genes. Nucleic acids can store and transmit genetic information from one generation to the next, fundamental to any life form.
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Transcribed Image Text:Exercise 3
The MTT assay is a widely used assay to study cell viability. It measures cellular metabolic
activity, which serves an indicator of cell viability, proliferation and cytotoxicity. This non-
radioactive colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals
by metabolically active cells. Viable cells contain NAD(P)H-dependent oxidoreductase
enzymes that reduce the MTT to formazan. The insoluble formazan crystals are dissolved and
the resulting coloured solution is quantified by measuring absorbance at 500-600nm using a
multi-well spectrophotometer. The darker the solution, the greater the number of viable,
metabolically active cells!
Exercise 3.1 MTT protocol
Your colleagues in the School of Biology and Environmental Science discovered an unusual
looking green algae on a recent fieldtrip to Costa Rica. They have provided you with some
extracts from the plant and you're excited to measure their effects on the growth of cancer
cells in vitro. You decide to perform an MTT assay on two prostate cell lines: PC3 (metastatic
prostate cancer) and PWR1E (immortalised non-cancerous epithelial cells). But "Oh sugar!"
your dog ate your lab book! Now you have to try and piece together the correct experimental
protocol, which has become all jumbled up.
Jumbled up protocol:
Incubate plate for 3h. Resuspend cell pellet in cell culture media. Add MTT reagent to each
well. Dissolve formazan product using DMSO. Count cells using haemocytometer. Prepare
desired concentration of compound for treatment. Seed appropriate number of cells in
each well of 96-well plate. Measure absorbance using plate reader. Add treatment to each
well of plate and incubate for 72h. Remove media and MTT reagent.
Correct experimental MTT protocol:
What would be the most appropriate controls to include in this experiment and why?
II
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