MTT assay was used to analyze cell viability of MCF 10A, MCF 7 and MDB-MB 231 cell lines in control and 10mM inorganic phosphate treated groups. MTT assay is a colorimetric assay for evaluating cell metabolic activity. MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) is a yellow aqueous tetrazolium dye. In living cells with active mitochondria, the mitochondrial enzymes such as succinate dehydrogenase (NADPH dependent oxidoreductase enzyme) reduce this dye to formazan, a purple-colored water-insoluble complex (Gomez et al., 1997). The intensity of absorption of the purple complex is read typically at 595nm using a spectrophotometer and is a direct measure of mitochondrial activity.
The materials and equipment required to perform this assay included MTT at 5mg/ml in calcium-free PBS (Cat #M 2128-1gm, Lot #MKBS4732V, Sigma Aldrich), stored at 40C in the dark, isopropanol (Cas # 67-63-0, J.T Baker, microcentrifuge tubes- 1ml (Catalog #211511, Lot #447425-Y23093, Avant, LPS, Rochester, NY), a 96-well plate for reading (Ref #353072, Falcon, Corning-Life Sciences, Durham, NC), pipette tips/repipettor (1ml, 200ul,Ref #702390, Eppendorf, Germany), centrifuge (Centrifuge 5415D, Eppendorf, Germany) and spectrophotometer (EL808 Microplate, BioTek Winooski, VT).
Samples were
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Silver nitrate solution (5%) was added (1ml/well) and cells incubated for 60min under a UV light source. At the end of 60 min exposure, the wells were rinsed with several changes of 1ml/well distilled deionized water. After this, 5% sodium thiosulfate was added (1ml/well) and cells were incubated at room temperature for 5min. This was followed by several rinses of distilled deionized water and the wells were photographed under phase contrast to see visible black/brown
The unknown bacterium that was handed out by the professor labeled “E19” was an irregular and raised shaped bacteria with a smooth texture and it had a white creamy color. The slant growth pattern was filiform and there was a turbid growth in the broth. After all the tests were complete and the results were compared the unknown bacterium was defined as Shigella sonnei. The results that narrowed it down the most were the gram stain, the lactose fermentation test, the citrate utilization test and the indole test. The results for each of the tests performed are listed in Table 1.1 below.
The isomerization procedure was done in order to create dimethyl fumarate from dimethyl maleate. Dimethyl maleate and dimethyl fumarate are cis and trans isomers, respectively. This procedure was done via a free radical mechanism using bromine. The analysis of carvones reaction was done in order to identify the smell and optical rotation of the carvone samples that were provided. The odor was determined by smelling the compound and the optical rotation was determined using a polarimeter.
Experiment: First prepared a well plate with the appropriate amounts of distilled water, HCl, and Na2S2O3 in each well according to the lab manual. The well where the reaction
Because it is a way of knowing the pressure that the blood is putting on the walls of arteries and veins.
coli cells were grown at 37 degrees Celsius with calcium ions. These cells were then incubated in ice and placed into 4 polypropylene tubes. Each tube consisted of 25 microliters of E.coli cells. Two of the four were labeled with (+) while the other two were labeled (-). Positive was with samples that contained 100 pg/ microliter of pGLO, while negative was for the samples that did not. The 4 tubes were incubated in ice for 30 minutes, then heat shocked for 30 seconds at 42 degrees Celsius. The 4 tubes were then returned to the ice bath for 5 minutes. After the removal, .975 microliters of LB media was added to each tube at room temperature. The tubes were then shook at 225 RPM at 37 degrees Celsius for one hour. 300 microliters of cells were then extracted from the tubes and then placed in agar plates. The cells were spread across the plate and the incubated overnight at 37 degrees
AGEs alter the mechanical properties of cells and tissues by crosslinking intracellular and extracellular proteins. They also bind to cell surface receptors called receptor for AGEs (RAGE), thus interrupting various cellular processes. Through laboratory experiments, scientists have shown that glycation of mitochondrial proteins, lipids and DNA may induce mitochondrial dysfunction due to a decrease in ATP production and increased free radical formation. The mitochondria are specialized...
The methods used for this lab came from Leady, B. (2014) Fundamentals of Life Science Lab Manual. Toledo, Ohio: University of Toledo. No changes were made.
The "Abstract" - "The. National Center for Biotechnology Information. U. S. National Library of Medicine, 31 May 2006. Web. The Web. The Web. 09 Apr. 2014.
Schulman, Joshua M., and David E. Fisher. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 28 Aug. 0005. Web. 24 Apr. 2014.
Aconitase catalyzes the conversion of citrate to isocitrate in the mitochondria and cytosol. In the mitochondria, aconitase is required for the TCA cycle to continue. In the case of high mitochondrial ROS production, aconitase becomes oxidized and no longer functions...
Wen, Chuck K., Pamela L. Hudak, and Stephen W. Hwang. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 06 Apr. 2007. Web. 13 Apr. 2014.
Enzymes are necessary for life to exist the way it does. Enzymes help our bodies carry out chemical reactions at the correct speed. Catalase is one such enzyme, “Catalase is a common enzyme found in nearly all living organisms exposed to oxygen (such as bacteria, plants, and animals). It catalyzes the decomposition of hydrogen peroxide to water and oxygen”.\(Wikipedia). In other words catalase speeds up the breaking down of hydrogen peroxide, which is a byproduct of reactions in our body. Hydrogen peroxide is very common in our body but, “If it were allowed to build up it would kill us”(Matthey).This shows how necessary enzymes such as catalase to life. Without enzymes reactions that take place in our body could be affected greatly. In our
The amino acids are stained purple and from this we can measure relative distance traveled (RF) to determine which amino acids are present in each sample. The more hydrophobic the amino acid the the further up the paper it appears this is why leucine is above alanine. Alanine is more hydrophobic than glutamate which is at the bottom of the paper chromatography.
== § Test tubes X 11 § 0.10 molar dm -3 Copper (II) Sulphate solution § distilled water § egg albumen from 3 eggs. § Syringe X 12 § colorimeter § tripod § 100ml beaker § Bunsen burner § test tube holder § safety glasses § gloves § test tube pen § test tube method = == = =
My hypothesis was I think that if we change the temperature of the enzyme it will have an area where the enzyme works very well, but if we increase or decrease the temperature too much, it will slow the productivity. If we change the pH of the enzyme, there will be an area where it is extremely productive, but if it gets too acidic or basic the productivity will slow. If we change the concentration by increasing the amount of enzymes, it will be more productive. T think this because your are having more than 1 enzyme work on one task. I you decrease the amount of enzymes, the productivity will be slower because there are less enzymes working on that certain task. The data refutes my hypothesis because I found that the enzymes work best in one