Cell Adhesion Practical - Term Paper Example

?Cell Adhesion Practical Introduction Cell adhesion assays can be used to explore cell-matrix interactions in several different types of cell, including epithelial and tumour cells, and thus assays are very important in all areas of biology and biomedical research. One of the most important areas of recent research using assays has been the investigation of cadherins, which are cell-cell adhesion molecules, dependent on Ca2+, which play an important role in ‘tissue construction and morphogenesis’ (Asano et al, 2004). Cadherins are found as trans-membrane proteins (Rastogi, 2007). There are four main types of classical cadherins, with the epithelial cadherin playing an important role in the growth of tumours (Asano et al, 2004). Cells possessing cadherins generally adhere to other cells possessing cadherins. By exploring these cell adhesion mechanisms with an assay, it is possible to understand more fully the role of the intracellular matrix and the reactions which occur within it. The assay described below relies on the interesting effects that trypsin has on these cadherin molecules. In the presence of calcium and trypsin, the cadherins are protected from cleavage, but in the presence of a trypsin and EDTA mixture, the cadherins are removed from the cell surface and thus the cells stop aggregation. Results Trypsin is commonly used to detach the adhering cells (Humphries, 2001). Prior to the addition of trypsin, the cells were adhering to each other forming a suspended clump of cells within the wells. However, after the trypsin was added, there was less evidence of adhesion and the cells were in the matrix singularly. After fixing, only the adhering cells were found in the well. This again gave a ‘clumped’ look to the cells. After viewing this, the wells were stained with methylene blue, and this the wells had a typical blue appearance in the top two rows, suggesting normal adhering cells were present. Sketch Cell Count Calculation The number of cells plated per well was calculated using a haemocytometer. Concentration of cells in original mixture = (A/(chamber size)(volume of chamber))(volume of sample dilution/volume of original mixture in sample) (Rastogi, 2007) In 1ml of cell suspension, there are A x 104 cells. The actual volume used in the well was 100µl which means that there was an actual value of (A x 104)/10, or A x 103 cells plated per well. Raw Data 1 2 3 4 5 6 7 8 9 10 11 12 A 0.935 0.685 0.798 0.857 2.35 2.323 2.253 2.523 2.427 2.809 2.58 2.416 B 0.445 0.488 0.632 0.401 2.165 2.13 2.308 1.708 0.774 0.851 1.072 0.814 C 0.028 0.023 0.037 0.028 0.025 0.025 0.025 0.025 0.026 0.025 0.026 0.027 D 0.029 0.031 0.027 0.029 0.028 0.023 0.029 0.026 0.027 0.026 0.035 0.03 E 0.027 0.033 0.028 0.03 0.022 0.013 0.029 0.028 0.032 0.022 0.032 0.03 F 0.028 0.024 0.026 0.028 0.027 0.028 0.022 0.028 0.017 0.014 0.021 0.027 G 0.028 0.032 0.024 0.026 0.037 0.021 0.029 0.017 0.018 0.016 0.024 0.034 H 0.032 0.019 0.025 0.023 0.025 0.014 0.028 0.014 0.018 0.013 0.022 0.025 Graph 1 A1-4 A5-8 A9-12 B1-4 B5-8 B9-12 Mean 0.819 2.362 2.558 0.492 2.078 0.878 Standard Dev. 0.105 0.115 0.183 0.100 0.258 0.133 Graph 2 A1-4 A5-8 A9-12 B1-4 B5-8 B9-12 Mean 0.201 0.370 0.346 0.158 0.325 0.407 Standard Dev. 0.009 0.023 0.067 0.020 0.031 0.021 Discussion ECM Component Preference As was previously discussed, cadhesins are of various different types and thus may have different preferences for the extra-cellular matrix in which they react. In the data above, the cells A1-4 represent matrix 1, A5-8 represent matrix 2, and A9-12 represent matrix 3. The cells B1-4 represent the BSA and B5-8 and B9-12 represent tube 1 and 2 respectively. In this case, the data obtained experimentally shows that there is a definite component preference for matrices 2 and 3, with the first one having a very low absorbance value and thus a very low amount of adhering cells. The sample data also show a similar trend, although the amount of adhering cells in matrix 1 are not so low as in the experimental data. There is another small difference in that the sample data suggests that there is an ECM component preference suggests that there is higher adherence in matrix 2, whereas the experimental data suggests that adherence is higher in matrix 3. The differences in the absorbance values appears to be very small and thus this does not suggest a major problem with the data. Integrins Integrins are also receptors that play a major role in cell attachment, whether this is between other cells or the extra-cellular matrix Integrins also play a major role in cell-cell signaling, being involved in both outside-in and inside-out signaling (Rastogi, 2007). As with adhesins, there are many different types of integrin which will have different preferences and activities depending on the cell type and the extra-cellular matrix components. Some cells may also possess more than one type of integrin embedded in the membrane, which means that there are some very interesting effects to investigate concerning integrins. As the role of the integrin shares many similarities with that of the cadhesin, a very similar experiment can be used to test the function. For example, it has been found that collagenase-3 (which cleaves type I collagen) has an effect on a type of integrin reaction known as alpha3beta1 due to the presence of collagen fragments generated. Taking the very same reaction, a simple substitution of similar volumes of integrins for cadhesins and colleganase-3 for trypsin would have the desired effect. This experiment would help illustrate both the adherence properties of integrins and the ability that colleganase-3 has to halt these reactions and produce single-cells within the matrix. From this experiment, it could also be found which type of cell has the strongest reaction to collagenase-3 by ensuring that different cell types are investigated, alongside different extra-cellular matrices. It has also been shown that mAb13 (a monoclonal antibody) recognizes only the integrin class I molecules (Akiyama et al, 1989). As such, another similar experiment could be conducted replacing mAb13 with the trypsin in the original experiment. This could again be used to change the patterns of cell adhesion and the results stained and measured with methylene blue. References Akiyama, S.K. et al., 1989. Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. The Journal of Cell Biology, 109(2), p.863 -875. Asano, K. et al., 2004. Correlation of N-cadherin expression in high grade gliomas with tissue invasion. Journal of Neuro-Oncology, 70(1), p.3-15. Humphries, M.J., 2001. Cell adhesion assays. Molecular Biotechnology, 18(1), p.57-61. Rastogi, S.C., 2007. Cell Biology, New Age International. Roskelley, C.D., Srebrow, A. & Bissell, M.J., 1995. A hierarchy of ECM-mediated signalling regulates tissue-specific gene expression. Current opinion in cell biology, 7(5), p.736-747. Read More