Cell Culture and Protein Detection

Cell Culture and Protein DetectionOverviewThis practical is split into three main break ups, each transgress allows us to develop certain techniques. The maiden part of the practical is focused on the techniques needed to successfully carry away cell cultures. The encourage part is using an analytical technique known as enzyme-linked-immunosorbent serologic assay this is used to measure the amount of proteins secreted by the cells that were cultured in part one. Lastly, part three focuses on Hesperian blotting, this is a technique used to measure proteins too but it differs from ELISA in that it measures proteins that be break inside the cell. The implicit in(p) objective of this practical is to look at how Bacterial lipopolysaccharide (LPS) alters the ripening and the look of the smooth muscle cell -actin (SMA) in vascular smooth muscle cells (vSMC) date also detecting and quantifying cell pointling molecules (JNKs) and cytokine secretion (TNF-) in vascular cells. clas s 1 SubcultureObjectiveThe aim of this part of the practical is to subculture the bovine aortic vascular smooth muscle cells and to count the cells and check for viability by counting the cells in suspension on a haemocytometer using a microscope. The cells are also toughened with a bacterial antigen (LPS) to look at its effect on viability, differentiation state and the activation of intracellular signalling and secretion of proteins. Immunocytochemistry is also carried out in this part of the practical.MethodTrypsin is used to remove the cells from the bottom of the flask. When the cells have dissociated, medium which contains a trypsin inhibitor is added. The medium stops excessive trypsin action from damaging the cells. The cell suspension is first diluted using trypthan blue to allow viable cells which remain white to be distinguished from non- viable cells which are blue. This method allows us to get a total cell count of cells/mL and the percentage of viable cells.Our total cell count was found to be 7.45105 cells/mLThe non-viable cells were found to be 1.5104 cells/mLThis means that the culture contains more 98% viable cells which indicates that it is a healthy culture.3 different 6 well casings were then set up using different densities of seed cells.3105cells/ml seeding constriction was used in one plate. This density was used as a lot of protein needs to be expose for the assessment of JNK and -actin protein cytokine yieldion.1105 cells/ml seeding density was used in a nonher 6 well plate. These plates are used to assess the effect of LPS on growth and viability of the cells. This density is used to give a clear picture of the cells to make counting easier.5103 cells/ml seeding density was used in the last plate. This plate is used for -actin expression by immunocytochemistry. The low density will give us a clearer picture.The three different sets of plates are all treated with varying concentrations of LPS. Each plate has two wells which are used as controls, containing no LPS, two wells containing 1g of LPS and two wells containing 10g of LPS.Immunocytochemistry is carried out on the plate with a seeding density of 5103cells/ml. This technique is used to determine if a particular protein or antigen is present. An unlabelled primary antibody is used to bind to the antigen sought after antigen. The presence of contractile protein -actin undersurface determine the differentiation state of vSMC. Immunocytochemistry is used as a qualitative method of determining the presence of a protein, it is not quantitative.ResultsCalculations for different seeding densitiesInitial concentration = 7.45105 cells/mLFormula = Plate 1, seeding density 1.5105 cells/mlPlate 2, seeding density 0.5105 cells/mlPlate 2, seeding density 2.5103 cells/ml slacken 1 Cell growth for each groupImmunochemistry tops fingerbreadth 1 Cell growth in the control (0g/ml LPS)Figure 1 Cell growth in the control (1g/ml LPS)Figure 3 Cell growth with 10g/ml of L PS word of honorThe first part of this section was to carry out a cell count and determine the viability of the suspension. Our assay had 7.45105 cells/ml and it contained more than 98% viable cells. This meant it was a healthy cell suspension and it was suitable to run tests on for the practical. A seeding density of 1105 cells/ml seeding density was used to assess the effect of LPS on growth and viability of the cells. This density was used to give a clear picture of the cells to make counting easier. The leave behinds of our look into correlated with the majority of the class. However there were some unheralded results in some of the groups but this could be down to plates being labelled in decryly or mistakes while counting when using the haemocytometer. Immunocytochemistry is used to allow us to analyse the results visually. We found that as the amount of LPS increased the number of cells decreased, this is illustrated in figures 1, 2 and 3. LPS is an endotoxin and it inhib its the growth of -actin. LPS also damages the structure of -actin, high levels of LPS stop the correct formation of the filaments and so affects the function of the cell which is to facilitate cell contraction and migration. This result was expected as LPS is a major mediator to septic stripe and is known to directly affect vascular smooth muscle cells.QuestionWhat could you do to improve this experiment?To improve this experiment I would use a wider range of concentrations for LPS. This would give a better understanding of its affects. Repeating the experiment several times and getting an average of your results would also help.Section 2 ELISAObjectiveThe aim of this section of the practical is to use Enzyme-Linked Immunosorbent Assay (ELISA) to detect production of the cytokine TNF from the cells activated with LPS. Detection is based on a food coloring change. Firstly a standard curve moldiness be generated so that absorbance shelters can be converted into concentrations of TNF-. Once the curve is completed we can determine the unknown concentrations of TNF- in our samples.MethodFirstly the antibody is immobilised onto the surface of the plate. The plate is then washed to remove any excess antibody, antigen is then added and it is allowed to bind to the antibody. A secondary antibody is then added, this antibody is labelled with an enzyme. The enzymes substrate is then added, this causes a colour change. The amount of coloured product formed is determined spectrophotometrically. The amount of coloured product is proportional to the amount of enzyme present and also to the concentration of the antigen.ResultsTable. 2 Absorbance values of samples at 450nm.Figure 4. Plot of standard curve of absorbance versus concentration.DiscussionThe aim of this experiment was to quantify the amount of TNF- present in our samples. To do this a set of known standards were used and their absorbance values read. This data produced a straight line with an R2 value of 0.99 indicating that a straight line was an excellent fit for absorbance versus concentration, and so the equation of the line could be used to determine unknown concentrations of TNF- based on their absorbance values. Our results showed that TNF- was present in its highest concentration of 25.15pg/ml in the sample with the highest concentration of LPS and it was found in its lowest concentration of 9.9 pg/ml in the sample containing no LPS. This result was expected as cytokines much(prenominal) as TNF- are produced in large quantities to respond to endotoxins such as LPS.QuestionWhat could you do to gain more learning from this experiment?To gain more information from this experiment you could test for other cytokines which are also activated by LPS, correlating these results would make your data more meaningful.Section 3 Western BlottingObjectiveThe aim of this section of the practical is to prepare cell lysates from the vascular smooth muscle cells which were activated by LPS previ ously. SDS PAGE and western blotting will then be used to detect the activation of the intracellular protein JNK. Western analysis quantifies the amount of protein present in the cell. To do this cell lysis must be carried out. In this practical we used a method which generates whole cell lysates. To do this lysis buffer is added to the cells followed by sonication.MethodCell lysis is carried out first. The undermentioned step is SDS polyacrylamide gel electrophoresis. Western blotting involves transferring the protein bands from an acrylamide gel to a more stable and immobilising medium such as nitrocotton paper so that analytical procedures such as detection with antibodies can be carried out. We carried out western blotting using iBlot dry blotting system. After blotting probing is carried out to determine the presence of phosphorylated JNK protein.ResultsFigure 5. Ponceau S StainingFigure 6. -actinFigure 7. pJNKDiscussionTo see if our transfer was a success before probing, the blot was stained with Ponceau S stain. The proteins can be seen as red bands with this stain. The result of this stain can be seen in figure 5. red bands are present which indicates our transfer was a success and that there are proteins present. The western blot analysis showed that -actin was present in all the samples as a strong band around 42kDa was observed which is expected for -actin. The results for pJNK did not go bad out as it was washed incorrectly, because of this no bands were observed, however bands would be expected in the samples containing LPS.Why did you run the sample on the gel before blotting?The sample was run on gel first as proteins are separated by molecular weight. This allows us to distinguish -actin from other proteins. It is then moved to the nitrocellulose paper so that analytical procedures such as detection with antibodies can be carried outWhy measure the phosphorylated form of JNK?The phosphorylated form of JNK is a signal a cell sends out when it is stressed. Therefore pJNK should be present in the samples with LPS. If it is present then it confirms the fact that the cell is stressed as a result of the presence of the endotoxin.