vitro cytotoxic study Biological activity for anti-cancer activity is created using the screening

After the serial dilutions of the red and blue dyes were taken, the molarity and absorbance for both dyes were calculated. Using the MiVi = MfVf equation, the concentrations for each value of the red and blue dye were separately calculated. Calculating absorbances calls for setting the correct wavelengths of light for each dye. In this case, the 470 nm wavelength for red dye and the 635 nm wavelength for blue dye was needed to find the maximum absorbances. The absorbance was found by blanking the colorimeter and entering the concentrations. After both values of the absorbances and concentrations were found, the values were then graphed in order to obtain the equation of the relationship between absorbance and concentration.

Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.

Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.

15) Obtain the boiled chloroplast suspension, mix, and transfer 3 drops to cuvette 4. Immediately cover and mix cuvette 4. Insert it into the spectrophotometer's sample holder, read the percentage transmittance, and record it in Table 4.4. Replace cuvette 4 into the incubation test tube rack. Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette's contents just prior to each readings. Remember to use cuvtte 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.

Norcantharidin (NCTD), the demethylated analogue of cantharidin, has been used to treat human cancers in China since 1984. It was recently found to be capable of inducing apoptosis in human colon carcinoma, hepatoma and glioblastoma cells by way of an elusive mechanism.1 Norcantatharidins and their analogues are synthetic anticancer agents which have been used in various different procedures for the inducing growth arrest and apoptosis of tumour cells and also provide therapeutic and adjuvant therapeutic applications in the treatment of cancer agents.2 This report will discuss the various different NCTD that are currently being studied, highlighting the synthesis of these NCTD and their various biological pathways that are taken in order to inhibit the growth of the correspondent cancer cell. In particular NCTD induced apoptosis of the hepatoma cells will be considered and also highlighting the different views with the synthesis of the particular NCTD and also the effectiveness of the methodology and its observations in response to other NCTD and their mechanism in attacking the hepatoma cell site.

PC3 (8×104  cells/well) and LNCaP (3×105  cells/well) cells were seeded in 6-well plates in 1.5  mL of complete growth media. Cells were treated with 2.5 μM of free sorafenib or an equivalent sorafenib concentration of SMA-Sor, 3 μM of free nilotinib or an equivalent nilotinib concentration of SMA-Nilo, DMSO or SMA for 48  h. Cell cycle distribution was assessed using propidium iodide staining, as previously described {Somers-Edgar, 2011 #82}. Samples were analysed using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and the proportion of cells in each of G0/G1-, S- and G2/M-phases were determined using CellQuest Pro software (BD Biosciences, San Jose, CA, USA).

Staining the cells with dye allows us to visualize the structures of the cell more precisely under a microscope.

Three wells where eliminated from the experiment, because a vehicle control was not needed for this experiment. Images where then taken of the 9 wells, with the use of phase-contrast microscope, these photos were labeled (T0). After the images where obtained, the well-plate was placed back into the tissue culture hood, and the wells where treated with different treatments, so the effects could be observed. In row “A” contained the HepG2 cells by themselves, no treatment was added. Row “B” contained the HepG2 cells and 4.5µl of Ceramide, which was labeled as the positive control, and finally in row “C” the wells contained our HepG2 cells and the compound Epigallocatechin Gallate. The well was then placed back into the cell culture incubator for 24 hours. Following the incubation, photos where taken using the same phase-contrast microscope and the same area of each scratch, these photos are labeled (Tf). The before and after photos of the scratch are then used to figure out the percent wound closure, which determines the cell migration. In order to calculate the percent closure, the following equation was used; Percent closure=width of Tf box/width of the T0

For each value shown we can determine that there is a strong correlation between drug concentration and % cell viability. This can tell us that as the drug concentration increases the % cell viability decreases. This shows that the chemotherapeutic agent is having the desired effect.

A 6-well plate containing pre-plated lung cancer cells (cell line H460) in 3mL of media was prepared for the purposes of this lab. Two wells were labeled NT (no treatment) and LPS (lipopolysaccharide). The well plate was tipped to pool the media to the base of the well. 30 μL of LPS was added to the LPS well, and swirled to mix its content. As the cell was incubated for 15 minutes, 225 μL of RIPA and 25 μL of protease inhibitor were added to a 1.5 mL tube, and kept on ice. Six 1.5 mL tubes were labeled NT-1, NT-2, NT-DIL, LPS-1, LPS-2 and LPS-DIL, and placed on ice. After 15 minutes all media was removed from the NT and LPS wells using the P1000 micropipette at 1000 μL. The remaining media was removed with the pipette by titling the plate allowing

15 cell lines were acquired for the purpose of this experiment. Process of subcellular compartment fractionation was performed on these cell lines, this included complete cells, nucleus and cytosol (seen in

In this experiment the purpose was to see if E. coli could express a his-6 tagged recombinant Green Fluorescent Protein (rGFP) in a bacterial culture. Purifying the sample through Ni 2+ Agarose Chromatography and then discovering the total protein yield through Bradford Assay determined expression of his6 tagged recombinant rGFP in E. coli. The purity of rGFP in the samples was examined and the strength of the bands that appeared at about 34.0 kDa (the MW of rGFP) in association to the ladder lane of the SDS-PAGE gel. Then a Western Blot was performed for the comparison of rGFP bands to the ladder to reasonably determine the molecular weight of rGFP. It was determined that the purity of the band of E2 was at 80 %, which made the calculated

After the location of bead placement is determined, one set of cells will be grown with the small molecule in heavy medium and another control set of cells in regular medium with no compound. After incubation, the proteins that have attached to the small molecule can be determined through mass spectroscopy will be

The breast cancer cells were incubated at 37 °C under normal atmospheric pressure, while the normal breast cancer cells were incubated at 37 °C in environments with 5% CO2 levels, respectively. The MDA-MB-231 and Hs578bst cells were grown respectively in L-15 or 46-X base media. These were supplemented with 100 I.U./ml penicillin/100 μg/ml streptomycin and 10% FBS. The cells were first rinsed with 1X PBS and then fixed with 4 wt% paraformaldehyde solution for 10 mins at room temperature. They were then rinsed with PBS solution for three more times, before a final rinse with purified water. Finally, the samples were dried in a vacuum desiccator for at least 5 hours.

Mitochondrial Membrane Potential Detection JC-1 Kit (BDTMMitoScreen) and FITC Annexin-V-Apoptosis Detection Kit I (BD PharmingenTM) were obtained from BD Biosciences. Lipofectamine® 2000, OptiMEM, and JC-1 (5,5’, 6,6’- tetrachloro-1,1’,3,3’tetraethylbenzimidazolocarbocyanine iodide) were purchased from Life Technologies and Molecular Probes respectively. TUNEL assay kit, Dual luciferase reporter gene assay kit and Proteasome-GloTM assay reagents were obtained from Promega. Anti-ubiquitin, anti-GFP, anti-20S proteasome, anti-p53, anti-p27, anti-p21, anti-Bax, anti-IkB-α, anti-β-actin and anti-HA antibodies were purchased from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated anti-rabbit-IgG and anti-mouse IgG; anti-rabbit, and anti-mouse (IgG fluorescein isothiocyanate and IgG-rhodamine) were obtained from Vector Laboratories. Plasmid pcDNA™ 3.1 was purchased from Life Technologies. pcDNA3-EGFP (Addgene 13031), pRK5-HA-Ubiquitin-WT (Addgene plasmid 17608), pEGFP-Hsp70 (Addgene plasmid 15215) and Luciferase-pcDNA3 (Addgene plasmid 18964) plasmids were purchased from Addgene.