Osmotic Pressure Effect on Plasma Membrane of Blood Cells

Osmotic Pressure feat on germ plasm Membrane of Blood CellsSyed IbrahimIntroductionThe plasma tissue layer is vital as it defines the boundary between cellular phoneular ph whizs and their environment (Heidcamp et al., 2014). Plasma membranes atomic turn 18 crucial in maintaining electrochemical gradients, controlling material exchange, and all(a)owing signal transduction (Zhao et al., 2004). The take of this lab was to identify isoosmotic solutions by examining the effect of osmotic pressure on the plasma membrane of line of credit cells and genus Elodea guard cells as intimately as to understand cell viability and membrane integrity using the Trypan colored ejection assay.To examine the effects of osmosis, fleshly railway line cells and guard cells from Elodea leaves were examined in this lab. Plasma membranes are found in both animate being cells and sic cells however, cell walls are notwithstanding present in plant cells (Freeman et al., 2011). It was pass j udgment that these cells would shrink in hypertonic solutions, stay the akin sizing in isotonic solutions, and swell (in the case of Elodea guard cells) or nevertheless burst (in the case of puppet blood cells) in hypotonic solutions (Heidcamp et al., 2014). Since glucose has a caravant Hoff reckon of 1 slice kilobyte chloride (KCl) has a vant Hoff constituent of 2, it was predicted that 0.15M glucose and 0.07M KCl would be the isoosmotic solutions for both the animal blood and the Elodea guard cells. Concentrations greater than the isoosmotic solution were expected to be hyperosmotic, succession tightfistednesss less than it were expected to be hypoosmotic.Viable cells exact sacrosanct plasma membranes (Heidcamp et al., 2014). In this lab macrophage cells, a type of leukocytes, were interact with variant abstruses and the effect of these compounds on the membrane of the cells was examined using Trypan forbidding, which differentially stains workable and non-viable c ells found on whether the cells ingest an intact plasma membrane (Freeman et al., 2011 Strober, 2011). Of the three know compounds, it was predicted that hepes-buffered RPMI (HPMI) would obturateage the membrane the least as it contains vitamins and supplements, and it was predicted that methyl group -cyclodextrin (MCD) would damage the cell the most as it removes cholesterol from plasma membranes (LifeTechnologies, 2013 Rodal et al., 1999).ResultsThe following three equations were utilise in calculations. Equation 1 was utilise to calculate os hoagyity, in read to determine the osmotic effect of a particular solution, (Heidcamp et al., 2014). Equation 2 was utilize to calculate osmotic pressure and Equation 3 was used to convert Celsius temperature to kelvin temperature (Heidcamp et al., 2014 USMA, 2012).Where = osmolarity = vant Hoff calculate = molar parsimony Where = temperature = temperature Where = osmotic pressure (kPa) = vant Hoff factor = molar concentration = ideal gas constant = (Chieh, 2002) = temperature Part A Lab 2 Report SheetsPlease refer to attached sheets.Part B Answers to delegate Questions ground on the observations of my colleagues and myself, 0.15M glucose and 0.15M cat valium chloride (KCl) were the isotonic solutions for the Eloda guard cells, plot 0.15M glucose and 0.035M potassium chloride (KCl) were the isotonic solutions for the animal blood cells.Solutions with solute concentrations greater than the isotonic solution were hypertonic, while concentrations less than it were hypotonic. Based on the observations from the lab, 0.6M glucose and 0.3M glucose were hypertonic for both the plant and blood cells, while 0.07M glucose and 0.035M glucose were hypotonic for the plant and blood cells. Additionally, 0.6M KCl, and 0.3M KCl, were hypertonic for the plant cell, while 0.07M KCl and 0.035M KCl were hypotonic for the plant cell. As well, 0.6M KCl, 0.3M KCl, 0.15M KCl, and 0.07M KCl were hypertonic for blood cells while on that point were no observed hypotonic KCl solutions for blood cells.It is expected that the isotonic solutions of glucose and KCl leave have the same osmolarity. Based on Equation 1, osmolarity is the product of the vant Hoff factor and molar concentration. Since glucose has a vant Hoff factor of 1 while potassium chloride (KCl) has a vant Hoff factor of 2, it is expected that glucose will have double the molar concentration of KCl to regain the same osmolarity in the isotonic solutions. The observations from this lab reveal that for plant cells, glucose and KCl had the same concentrations for isotonic solutions while for animal cells, glucose had approximately four times the molar concentration of KCl for isotonic solutions. This may have occurred due to the subjective character of classifying tonicity, and since these observations were split up amongst four groups, their subjective impressions may have been different. perfectly, one group to do all four sets, but due to tra mmel time in the lab, this was not possible.Tween-20 is a non-ionic type of surfactant that burn down be used solubilizing agent for membrane proteins (Iwahashi et al., 1991 Sigma, 2003). Bovine serum white (BSA) is a protein that adsorbs onto negatively charged portions of the membrane bilayer, forming temporary gaps in the membrane, therefrom increasing the permeableness of the membrane (Tsunoda et al., 2001). Based on this information, Tween-20 would liable(predicate) attain to a greater extent damage on the membrane bilayer, resulting in more depressed cells (after the Trypan blue exclusion test). Therefore, the odd un cognize is most likely Tween-20, and the evening unknown is most likely BSA. search locomotive Web of intelligence inquisition wrong Tween 20 sorted by relevanceReference Iwahashi, K., Tsubaki, M., Miyatake, A., Miura, S., Hosokawa, K., Ichikawa, Y. (1991). Catalytic properties of cytochrome P-450scc from bovid and porcine adrenocortical mitochondria Effect of tween20 concentration. The Journal of Steroid Biochemistry and Molecular biota, 38(6), 727-731.Search Engine GoogleSearch cost Tween-20Reference Sigma. (2003). Tween-20 (P5927) ingathering Information. SigmaAldrich. Retrieved October 6, 2014, from https//www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/p5927pis.pdfSearch Engine Web of ScienceSearch Terms bovine serum albumen sorted by relevanceReference Tsunoda, T., Imura, T., Kadota, M., Yamazaki, T., Yamauchi, H., Kwon, K. O., et al. (2001). Effects of muramidase and bovine serum albumin on membrane characteristics of dipalmitoylphosphatidylglycerol liposomes. Colloids and Surfaces B Biointerfaces, 20(2), 155-163.Based on the Trypan blue exclusion assay observations recorded in put over 2.2, unknown 1 (most likely Tween-20) caused the most damage to the plasma membrane as it had the highest function of blue cells (60%). Tween-20 belongs to the class of polyoxyethylene sorbate com bounds (Boxtel et al., 1990). Among some functions, these compounds solubilize the proteins and lipids found in the plasma membranes, thereby lysing the membranes (Boxtel et al., 1990). This allows Trypan blue to scratch the lysed membranes, making them blue.Discussion and Conclusionssummary of findingsIn the first part of this lab, the effects of osmosis were examined on animal blood cells and Elodea guard cells. It was determined that isoosmotic solutions for the Eloda guard cells were 0.15M glucose and 0.15M potassium chloride (KCl), while the isoosmotic solutions for the animal blood cells were 0.15M glucose and 0.035M KCl. Solutions with solute concentrations greater than the isotonic solution were hypertonic, while concentrations less than it were hypotonic. It was expected that the isotonic solutions of glucose and KCl would have the same osmolarity, since neither of them can easily scatter across the membrane without the use of a channel or head protein (Heidcamp et al., 2014) . Based on Equation 1, osmolarity is the product of the vant Hoff factor and molar concentration. Since glucose has a vant Hoff factor of 1 while potassium chloride (KCl) has a vant Hoff factor of 2, it was expected that glucose would have double the molar concentration of KCl to obtain the same osmolarity in the isotonic solutions. The results from this lab do not reflect this theoretical expectation. This may have occurred due to the subjective nature of classifying tonicity. Ideally, one group should to do all four sets of observations for Table 2.1, but due to limited time in the lab, this was not possible.In the second part of this lab, membrane integrity and permeableness was examined using the Trypan blue exclusion. Normally Trypan blue is not permeable in living cells. However, if the plasma membrane is not intact, Trypan blue is able to enter the cell, staining it blue (Heidcamp et al., 2014 Strober, 2011). Once treated with the specific compounds (such as genus Gly cine or Tween-20), the Trypan blue exclusion assay was performed on the RAW macrophage cells. Afterwards, the number of blue (non-viable) and non-blue (viable) cells were counted and their respective percentages were calculated. Glycine is smallest of the twenty common amino acids, and some all (97%) of treated cell remained viable (Freeman et al., 2011). Methyl -cyclodextrin (MCD) is a compound that removes cholesterol from plasma membranes, making the membrane more permeable, and it resulted in only 55% viable cells (Rodal et al., 1999). Hepes-buffered RPMI (HPMI) is a medium that contains vitamins and supplements that can be used for cell growth, and nearly all (98%) of treated cells remained viable (LifeTechnologies, 2013). In gain to these three compounds, the cells were treated with two unknowns. The odd unknown resulted in comparatively few (40%) viable cells, while the even unknown resulted in many an(prenominal) (85%) viable cells. Tween-20 belongs to a class of purifyi ngs which are known to solubilize membrane proteins, thereby lysing the membranes (Boxtel et al., 1990 Iwahashi et al., 1991). Bovine serum albumin (BSA) is a protein that forms temporary gaps in the membrane, and so increasing the permeableness of the membrane (Tsunoda et al., 2001). Based on the literature, Tween-20 would cause more damage on the plasma membrane resulting in fewer viable cells. Therefore, the odd unknown was determined to be most likely Tween-20, and the even unknown was most likely BSA.Answer to questionsAn organism that thrives in high-salt environments is known as a halophile, such as Halomonas meridian (James et al., 1990 Ventosa, 1998).Search Engine Web of ScienceSearch Terms halophileReference James, S., Dobson, S., Franzmann, P., Mcmeekin, T. (1990). Halomonas meridiana, a New Species of Extremely Halotolerant Bacteria Isolated from Antarctic Saline Lakes. organized and Applied Microbiology, 13(3), 270-278.Search Engine Web of ScienceSearch Terms haloph ileReference Ventosa, A., Nieto, J., Oren, A. (1998). biology of Moderately Halophilic Aerobic Bacteria. Microbiology and Molecular biota Reviews, 62(2), 504-544.Methyl -cyclodextrin (MCD) is a compound that removes cholesterol from plasma membranes (Rodal et al., 1999). Thus it is expected, that MCD makes the plasma membrane more permeable, and allows Trypan blue to enter the cell. The results from this lab agree with this, as a relatively large percentage (45%) of cells treated with MCD was stained blue in the Trypan blue exclusion assay.Search Engine Web of ScienceSearch Terms methyl beta cyclodextrinReference Rodal, S. K., Skretting, G., Garred, O., Vilhardt, F., Deurs, B. V., Sandvig, K. (1999). Extraction of Cholesterol with Methyl-beta -Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles. Molecular Biology of the Cell, 10(4), 961-974.The predictions are belowAcetone is an organic solvent that solubilizes lipids from the plasma membrane (Jamur Oliver, 201 0). High concentrations of acetone can disrupt lipid packing in the membrane, thereby increasing membrane fluidity and permeability (Posokhov Kyrychenko, 2013). Therefore is predicted that acetone will increase cell permeability and allow some of the Trypan blue into the cell, staining some cells blue.Methanol is an organic solvent that dissolves lipids from the plasma membrane (Jamur et al., 2010). Methanol can increase the rotational mobility of membrane lipids, increasing its fluidity (Joo et al., 2012). Similar to acetone, it is expected that methanol will increase cell permeability and allow some of the Trypan blue into the cell, staining some cells blue.Saponin is a detergent that selectively removes cholesterol from the plasma membrane, resulting in small holes in the membrane (Jamur et al., 2010). This would act in a manner similar to MCD, likely causing the cell to be permeable to Trypan blue and staining many cells blue.Triton X-100 is a non-ionic detergent that non-sel ectively solubilizes proteins from the plasma membrane (Jamur et al., 2010). This would act in a manner similar to Tween-20, likely causing the cell to be highly permeable to Trypan blue and staining a large percentage of them blue.Search Engine PubMedSearch Terms cell membrane permeabilityReference Jamur, M. C., Oliver, C. (2010). Permeabilization of cell membranes. Immunocytochemical Methods and Protocols, 588, 63-68.Search Engine Web of ScienceSearch Terms effect of acetone on membranesReference Posokhov, Y. O., Kyrychenko, A. (2013). Effect of acetone accumulation on expression and dynamics of lipid membranes studied by molecular dynamics simulations. computational Biology and Chemistry, 46, 23-31.ReferencesAlberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (2008).Molecular Biology of the Cell(5th ed.). New York miscellanea Science.Boxtel, R. M., Lambrecht, R. S., Collins, M. T. (1990). Effect of polyoxyethylene sorbate compounds (Tweens) on colonial mo rphology, growth, and ultrastructure of Mycobacterium paratuberculosis. Apmis, 98(7-12), 901-908.Chieh, C. (2002). The Ideal Gas Law. Ideal Gas. Retrieved October 5, 2014, from http//www.science.uwaterloo.ca/cchieh/cact/c120/idealgas.htmlFreeman, S., Harrington, M., Sharp, J. (2011). Biological Science (Canadian ed.). Toronto Pearson Canada.Heidcamp, W., Antonescu, C., Botelho, R., Victorio-Walz, L. (2014).Laboratory Manual Cell Biology BLG311(Fall 2014 ed.). Toronto Ryerson University.Iwahashi, K., Tsubaki, M., Miyatake, A., Miura, S., Hosokawa, K., Ichikawa, Y. (1991). Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria Effect of tween20 concentration. The Journal of Steroid Biochemistry and Molecular Biology, 38(6), 727-731.James, S., Dobson, S., Franzmann, P., Mcmeekin, T. (1990). Halomonas meridiana, a New Species of Extremely Halotolerant Bacteria Isolated from Antarctic Saline Lakes. systematic and Applied Microbiology, 13(3), 270-278.Jamur, M. C., Oliver, C. (2010). Permeabilization of cell membranes. Immunocytochemical Methods and Protocols, 588, 63-68.Joo, H., Jang, H., Yun, I., Bae, S., Chung, I., Bae, M., et al. (2012). The Effect of Methanol on the Structural Parameters of neuronal Membrane Lipid Bilayers. The Korean Journal of Physiology Pharmacology, 16(4), 255.LifeTechnologies. (2013). RPMI 1640 Medium, HEPES. Cell Culture Transfection Reagents. Retrieved October 6, 2014, from http//www.lifetechnologies.com/ high society/catalog/product/22400089productDetailPagePosokhov, Y. O., Kyrychenko, A. (2013). Effect of acetone accumulation on structure and dynamics of lipid membranes studied by molecular dynamics simulations. Computational Biology and Chemistry, 46, 23-31.Rodal, S. K., Skretting, G., Garred, O., Vilhardt, F., Deurs, B. V., Sandvig, K. (1999). Extraction of Cholesterol with Methyl-beta -Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles. Molecular Biology of the C ell, 10(4), 961-974.Sigma. (2003). Tween-20 (P5927) Product Information. SigmaAldrich. Retrieved October 6, 2014, from https//www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/p5927pis.pdfStrober, W. (2011). Trypan blue exclusion test of cell viability. Current Protocols in Immunology, 21(3B), A.3B.1A.3B.2.Tsunoda, T., Imura, T., Kadota, M., Yamazaki, T., Yamauchi, H., Kwon, K. O., et al. (2001). Effects of lysozyme and bovine serum albumin on membrane characteristics of dipalmitoylphosphatidylglycerol liposomes. Colloids and Surfaces B Biointerfaces, 20(2), 155-163.USMA. (2012). Metric System Temperature. U.S. Metric Association. Retrieved October 6, 2014, from http//lamar.colostate.edu/hillger/temps.htmVentosa, A., Nieto, J., Oren, A. (1998). Biology of Moderately Halophilic Aerobic Bacteria. Microbiology and Molecular Biology Reviews, 62(2), 504-544.Zhao, Y., Zhang, W., Kho, Y., Zhao, Y. (2004). Proteomic Analysis of Integral Plasma Membrane Proteins. Analytical Chemistry, 76(7), 1817-1823.