Identifying cells using Immunofluorscence Rachel Scalzo Bio-300, 001 Professor Bentley 9/24/2014

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1 Identifying cells using Immunofluorscence Rachel Scalzo Bio-300, 001 Professor Bentley 9/24/2014

2 2 Abstract In this experiment, we used immunofluorescence to isolate certain organelles and see where they were located within cells from mouse fiberglass. The organelles that were targeted were the nucleus, mitochondria, lysosomes, cytoskeleton, and the cell membrane. We used several dyes including: DAPI (which dyes the nucleus), Mito- Tracker (which stains the mitochondria), LysoTracker (which isolates the lysosome), and phallotoxin (stains the actin filaments within the cytoskeleton). Cells from mouse fiberglass were cultured and isolated into eight different wells. Each well was treated with a different dye, or combination of dyes except well #8, which was used as a negative control. The treated cells were kept in incubation for a week and were then viewed on a microscope that was able to stimulate the dyes using different colored lights. Despite the small error that was made during the procedure with wells #6 and #7, the overall pictures of the cell were very clear, and we were able to distinguish specific organelles using the dyes and determine that the cells were eukaryotic cells. Introduction When Robert Hooke discovered these chambers in cork that he dubbed cells, scientists have always been fascinated by what components make up a cell. As technology and scientific advancements have been made, scientists have been able to see wh at makes up the cell in great detail using a variety of different microscopes, computers, dyes, and other machines. One technique is called immunofluorescence microscopy, which is using fluorescent dyes to highlight specific organelles within the cell, then stimulating the dyes using different colored light. Immunofluorescence has been used to identify different characteristics of cells, such as characterizing different mammalian epithelial cells (2). Scientists have discovered over the years that cells have organs (or organelles) that have specific functions within the cell. One of the biggest organelles is the nucleus, which houses the genetic information of the cell. During this experiment, we used a dye called DAPI (which stands for 4 6- diaminidino-2-phenylindole) to stain the nucleus. When stimulated by colored light, it glows blue underneath the microscope. The lysosomes take care of disposing cell waste from inside the cell, as well as destroying organelles within the cell that do not function correctly. Due to the fact that lysosomes are constantly breaking down nutrients, their ph is very acidic. Since lysosomes are highly acidic in comparison to the rest of the cell, a dye called LysoTracker is used to stain the lysosome. LysoTracker is attached to a weak base, which is able to neutralize the acidic content with the lysosome and dye the lysosome. The mitochondria are often called the power house of the cell due to the fact that this is where ATP is formed via cellular respiration. There are hundreds of mitochondria within a typical cell due to the cell s constant need for energy. The mitochondria has two membranes where cellular respiration takes place, as well as a space between the two membranes that has an electron transport chain (ETC). The dye to stain the mitochondria, called Mito-Tracker, is able to mix with the ETC and stain the organelle within the cell.

3 3 The cytoskeleton is not only the structural support of the cell, it also is a transport highway that allows nutrients to get from one organelle to another. It is composed of actin, microtubules, and intermediate filaments. The cytoskeleton allows the cell to retain (or change) its shape, and to move from location to location. The dye used to stain the cytoskeleton is called phallotoxin, which targeted the actin within the cytoskeleton, making it glow red when stimulated with colored light. The cell membrane is made up of a phospholipid bilayer, which creates a boundary between the inside of the cell, and the outside environment. During this experiment, we stained the cell membrane to get a better idea of a cell s boundary s and overall size. The hypothesis of this experiment is to use fluorescence to determine what characteristics the cell has to identify what kind of cell it (whether it s a prokaryotic or eukaryotic cell). Materials & Methods The materials and procedure for this experiment are found on the American University Blackboard website under the contents folder for Cell-Biology-300 (1). The materials for this experiment consist of a slide of mouse fiberglass cells within eight wells, a 200 µl pipetman, Mito-Tracker, LysoTracker, DAPI, Triton X-100, PBS, and phallotoxin. In the first half of the experiment the liquid was removed from wells #1,3,4,6, and µl of MitoTracker was added into wells #1 and #7 and left alone for 20 minutes. Simultaneously, 100 µl of LysoTracker was added to well #4 and incubated for 20 minutes. 100 µl of plasma membrane dye was added to wells #3 and #6 and incubated for 20 minutes. After twenty minutes had passed, the Mito-Tracker, LysoTracker, and plasma membrane dye was removed from wells #1,3,4,6, and 7 and then immediately was washed with 100µl of PBS mixed with 4% paraformaldehyde. After the cells were washed with PBS, they were incubated for 15 minutes. Simultaneously, the liquid in wells #2,5, and 8 was removed and they were washed with 100 µl of PBS twice. After the second washing of PBS, 100 µl of 4% paraformaldehyde and PBS were inserted into wells #2,5,and 8 and incubated for 10 minutes. At this point in the experiment, all of the incubation times finished within a minute or two of each other so all of the paraformaldehyde was removed from each well. Each well was then washed with 100 µl of PBS, removed, and then 100 µl of more PBS was added to all the wells. However, following the second addition of PBS, all of the wells had the PBS removed except for wells #3, and #6. Wells #1,2,4,5,6, and 8 had 100 µl of Triton X-100 added, and those wells were left alone for 5 minutes. After five minutes had passed, the Triton X-100 was removed from wells #1,2,4,5,6, and 8 and had 100 µl of PBS added to wash them. The PBS was then removed from all of the wells and the final additions of the specific dyes were added to the wells. Well #1, #3, #4, and #8 had 100 µl of PBS added to them. Well #2, and #5 had 100 µl of phallotoxin added, as well as well #5 had 100 µl of DAPI added to it. Well #7 also had 100 µl of DAPI inserted inside of it. After incubating all of the wells for 20 minutes, each of the wells had their respective dyes and media removed, and had 100 µl

4 4 of PBS added and then the PBS was removed. This process with the PBS was repeated again to ensure the cells were washed. The plastic wells were then removed from the slide and 100 µl of glycerol in PBS was added to a cover slip and inserted on top of the slide. Any excess glycerol was wiped away with a Kimwipe and the cover slip was then coated with clear nail polish to ensure a tight seal. The slide was then incubated for a week at 4 C, after which we looked at our cells through a microscope and took photos with a camera on top of the microscope. The cells were stimulated with red, blue, or green light to show the fluorescence of the dye inside the organelles. Results Fig. 1 The cells in figure 1 have been stained with DAPI and phallotoxin, highlighting the nuclei and actin within the cells. This took place within well #5. The nuclei are the blue circles that are interspersed throughout the cells, while the actin is shown by the green lines and streaks that are in the photo. There appears to be more actin and nuclei on the right side of the figure than the left, which means that there are more cells towards that region of the photograph than at the bottom of the picture.

There are also green circles dispersed throughout the figure in the top right corner which is probably cell junk, or cell waste. Fig.

5 5 Fig.2 Figure 2 illustrates the nuclei of the cell, as well as the cell membranes of the cells. This took place in well #6. The nuclei are shown as bright, blue circles, while the cell membrane is the faded green surrounding the nuclei. The green regions around the nuclei allow us to estimate the size of the cells. There are also green circles dispersed throughout the figure in the top right corner which is probably cell junk, or cell waste. Fig. 3 Figure 3 depicts the nuclei and the lysosomes stained with DAPI and LysoTracker respectively. This took place in well #4. The nuclei of the cells are the blue circles, while the red stain represents the lysosomes. There is more red stain in the figure than blue because, while the nucleus is bigger than a ribosome, there are more lysosomes in a cell than one nucleus. Some of the red stain can also be residual cell waste that has been expelled from the cell after being absorbed by the lysosomes.

6 6 Fig. 4 Figure 4 shows the nuclei and the mitochondria stained with DAPI and MitoTracker respectively. This occurred in well #7. The blue circles represent the nuclei, while the little red dots that surround the nuclei represent the mitochondria. There are hundreds of mitochondria compared to the one giant nucleus. It appears that the brighter mitochondria are closer to the nucleus, while the mitochondria the furthest away from the nuclei are not as stained by the Mito-Tracker. Discussion During our experiment, the procedure was not followed according to the parameters within the document on the American University website. Towards the end of the experiment, 100 µl of Triton X-100 was inserted into well #6 instead of well #7. This ultimately caused the cells to become more permeable, allowing more substances to enter and leave the cell. The purpose of the Triton X-100 in this experiment was for the DAPI and Mito-Tracker to get into the cell easier, and ultimately have a brighter fluorescence. However, this mix up caused the cells in well #7 not to have a bright fluorescence, and it was harder to distinguish the different organelles because they were not stained properly. The other error that occurred was placing too much nail polish at the end of the experiment, which might have caused some of the nail polish to get on the slide, or a blurred picture. There is always human error that happened during the experiment, whether it was leaving the cells incubating a minute or two too long, or incorrectly measuring the contents of solutions that were going in the cells. The shape of mitochondria compared to the shape of a lysosome is very different from one another. Mitochondria are oval shaped, and are very thin with two membranes. However, lysosomes are more circular, and have a wider diameter than mitochondria. This is due to their different functions locations within the cell. Lysosomes have a wider diameter because they are absorbing more nutrients and storing cell waste for the entire cell, so they have to be bigger than some other organelles, like mitochondria. However, mitochondria are smaller because they are more efficient and need to be pretty small for cellular respiration to take place. The components needed for cellular respiration are so tiny that it would be unrealistic for them to be huge.

7 7 Actin is a one of the components that make up the cytoskeleton. It s more flexible than the other components of the cytoskeleton, and is partially responsible for movement, such as muscle movement in our muscle cells. Actin has four different locations during different phases of a cell s life. During interphase (or the majority of the time) actin is seen as long strands that stretch throughout the cell. However, during mitosis the actin disappears until cytokinesis where the actin is in the cleavage furrow to help it divide the cell into two (3). Negative controls are necessary in the majority of experiments to ensure that the absence of an independent variable will not change anything or cause anything to occur. The expected data within a negative control is for there to be no reaction. During this experiment, our negative control was in well #8, which did not have any special stain administered to it except getting washed several times with PBS. The control told us that without the dyes, none of the organelles could be seen. The cell in well #8 was clear, and very hard to see since the light just shown through the cell. Based on the photos taken of the cells, it can be concluded that these cells were eukaryotic cell due to the presence of major organelles such as a nucleus, lysosomes, and mitochondria. The dyes were able to show us the organelles that would not be present in prokaryotic cells (such as a nucleus), as well as the overall size of the cells. The cell membrane stain showed us that the cells were fairly large in comparison to your typical prokaryotic cell. Immunofluorescence can be used to identify numerous types of cells and the characteristics inside the cell that would not normally be visible to the naked eye. It gives an accurate representation of where organelles are placed within the cell as well as the number of specific organelles within the cell. For example, biology textbooks often show one or two mitochondria within a eukaryotic cell, when in reality, with immunofluorescence we can see that there are actually hundreds inside and that they re located near the nucleus. The way immunofluorescence targets specific organelles, I wonder if it is possible to use it to target specific pathogens, or viruses. If it were possible, how complex would it be to design a dye to target a virus (since it is not a cell, but has the characteristics of one)? From there, scientists could be on their way to cure illness such as AIDS, or even the common cold. That kind of discovery would revolutionize the world and change everything. References 1. Bentley Meg. (2014). Analysis of Cellular Organelles by Immunofluorescence. Biology 300 Cell Biology. Pp.1-2. Washington: American University. 2. Werner, F; Beate, A; Schmid, E; Freudenstein, C; Osborn, M; Weber, K. Identification and Characterization of Epithelial Cells in Mammalian Tissues by Immunofluorescence Microscopy Using Antibodies to Prekeratin 31, July, Differentiation. (Retrieved 23 September 2014) 3. Sanger, J.W. Changing Patterns of Actin Localization During Cell Division. Proceedings of the National Academy of Sciences of the United States of America. 72(5). Pp Retrieved 23, Sept

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Introduction to Cells

Life Science Introduction to Cells All life forms on our planet are made up of cells. In ALL organisms, cells have the same basic structure. The scientist Robert Hooke was the first to see cells under