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Response to extracellular stimuli lab report intro

Submitted by oringham on Thu, 04/05/2018 - 16:12

It is necessary for cells to be able to communicate at both intracellular and extracellular levels in order to grow and survive in a given environment. This laboratory exercise investigates the mechanisms of cellular response to internal and external stimuli, with respect to calcium ion transport, using a variety of agents (ATP, bradykinin, vasopressin). Exploration to determine whether a particular stimulus mediates calcium transport via plasma membrane channels or via release from intracellular stores was conducted using ionomycin (a calcium ionophore) to gain further insight on both levels of cellular communication.

Dog Persuasion Intro

Submitted by oringham on Tue, 04/03/2018 - 12:02

The choice of the German Shepherd as a breed of dog to receive the vaccine and be saved from extinction is the most logical and beneficial choice for mankind. German Shepherds have served as protectors and companions of humans for centuries, and have proven to be valuable assets to people across various and diverse lifestyles.

 

Conclusion Endocytosis Report

Submitted by oringham on Tue, 04/03/2018 - 11:59

Overall, this laboratory exercise emphasizes the similarities and differences of receptor mediated and non-specific endocytosis, and their significance in mediating transport of extracellular cargo. Additionally, the impact of other cellular machinery (i.e microtubules) on the efficacy of endosome migration is explored, highlighting the intricate collaborative nature of the cell. 

Discussion paragraph 3 477H

Submitted by oringham on Thu, 03/29/2018 - 16:11

Differences in vesicle movement across treatment and control groups in LLC-Pk1 cells can be explained by nocodazole’s depolymerizing effect on tubulin structure. In the control group, the integrity of the microtubule structure remained intact, enabling vesicles to bind their receptors to microtubule tracks. This then allowed for motor proteins to hydrolyze ATP, producing movement across the microtubule. Though much of this movement is similar in displacement, the data indicates an outlier. This outlier however was still chosen to be presented to exemplify the jerky nature of endosome saltation movement across microtubules, due to occasional detachments of endosome to microtubule. Nocodazole treated cells exhibited low average displacement of vesicles due to the depolymerization of tubulin, which did not allow receptors on the vesicle surface to bind and travel across tubulin structures. Because of this, endosomes remained suspended in the luminal space of the cell, slowly migrating to lysosomes in the interior to be degraded.

Discussion Paragraph 2

Submitted by oringham on Wed, 03/28/2018 - 23:32

Although quantitative analysis was not possible for fixed NIH 3T3 cells treated with TMR-dextran and lysotracker. This could have occurred due to possible insufficient exposure time and binning combinations when capturing images, or insufficient incubation time and conditions for the lysotracker and TMR-dextran. However, based on previous studies and common science knowledge, we are able to infer what visual and quantitative data of this investigation should resemble. After 15 min. of incubation time, the average total intensity of the TMR-dextran should be high at the surface of the cell relative to the interior. This is due to the relative brevity of the incubation time, where has not been sufficient time for many of the dextran molecules to be invaginated into the cell’s luminal space. The interior images should also display a relative high intensity of lysotracker at this point. After 75 min. of incubation time, the average total intensity of the TMR-dextran should be high at the interior of the cell relative to the surface. This is because the cell has had sufficient time to invaginate a majority of the dextran molecules and form endosomes and protonate, bringing them into the cell’s luminal space and closer to lysosomes. At this point, the interior images should display high lysotracker total intensities and high dextran total intensities, which would confirm that the dextran migrated to the inner most part of the luminal space.  

Discussion Paragraph 1

Submitted by oringham on Wed, 03/28/2018 - 23:32

At t=0 min., the average total fluorescence intensity of NIH 3T3 cells at the surface of the cell was relatively high due to the lack of time that the endosomes had to migrate to the interior of the cell. At t= 0, transferrin had just been introduced to the cellular environment, and were only able to quickly to bind to the receptors of the cell, which rest on the cell surface. After 30 min. of incubation, cells exhibited relatively similar average total fluorescence intensities on the surface and interior, demonstrating moderate amounts of migration of endosomes to the interior of the cell. This added time allowed for the ligand bound transferrin to be internalized via clathrin coated pits to coated vesicles and migrate deeper into the cell. After 90 min. of incubation, the interior of the cell exhibits a relative higher average total intensity compared to the surface. This again is due to the added incubation time, which has allowed the previously early endosomes to acidify, releasing iron, which then brings the transferring molecules closer to the interior where sorting endosomes are found. 

Results paragraph 2 endocytosis lab

Submitted by oringham on Wed, 03/28/2018 - 23:30

Live LLC-Pk1 cells with GFP tagged tubulin were treated with TMR-dextran. Control group cells were incubated with solely TMR-dextran, while treatment group cells were incubated with TMR-dextran, and subsequently incubated in nocodazole. Both groups were time-lapse imaged at 40X magnification for 300 s (1 frame every 10 s). Vesicle movement was then quantitatively analyzed using ImageJ’s “TrackMate” plugin to measure total displacement of vesicles (Figure 5). Cells treated with nocodazole demonstrated a lower average displacement of vesicles compared to control group cells (0.164µm, 0.716µm, respectively) (Table 1). Composite image frames of start, middle, and end of the time-lapse image series visually display the differences in movement of the vesicles over time across groups (Figure 4).

Results paragraph 1 endocytosis lab

Submitted by oringham on Tue, 03/27/2018 - 17:50

Surface and interior images of fixed NIH 3T3 cells treated with fluorescently labeled transferrin and lysotracker were taken at 100X magnification at three separate time points (0 min. incubation, 30 min. incubation, 90 min. incubation) in order to capture endosome migration over time (Figure 1). At t=0 min., it is clear that there is an abundance of transferrin molecules at the surface of the cell (Figure 1A), resulting in a relative high average total fluorescence intensity compared to the interior of the cell (2.5, 2.1 respectively) (Figure 2). At t=30 min. the average total fluorescence intensity of the surface and interior of the cell is relatively similar (3.3, 3.2, respectively) (Figure 2), At t= 90 min. there is a relatively higher average total fluorescence intensity at the interior of the cell compared to the surface of the cell (4.1, 3.3, respectively) (Figure 2).  Additionally, it can be visually noted that there is a significantly larger presence of transferrin molecules in the interior image than the surface image (Figure 1H, 1G, respectively). Lysozymes are unable to be visualized due to overpowering green stain in composite images. 3D projections of the cells were unable to be created due to immense technical difficulties.

Introduction Endocytosis Lab

Submitted by oringham on Tue, 03/27/2018 - 17:49

The process by which cells intake extracellular material from the environment is known as endocytosis, and can occur in both a receptor-mediated and non-specific manner. This laboratory exercise investigates both of these endpcytotic processes over time using fluorescence microscopy to observe endosome migration in fibroblast (NIH 3T3) and epithelial (LLC Pk1) cells treated with transferrin and TMR-dextran. Additionally, effects of nocodazole in LLC Pk1 cells was studied to explore the efficacy of vesicle motility along microtubules in cells with depolymerized tubulin.

Variation in Photosynthetic Levels due to Variation in Chloroplast Concentration Results Paragraph

Submitted by oringham on Wed, 03/21/2018 - 19:02

Tables 1 and 2 both numerically display the results of the spectrophotometer for both the spinach and romaine lettuce chloroplasts at each time interval and for each experimental group. Figure 1 displays an absorbance versus time graph for the romaine lettuce chloroplast reactions. It is apparent that the chloroplast and light group has the highest absorbance value by the end of the 30 minutes, while chloroplast + dark has the lowest absorbance. The chloroplast + light group also had the largest slope at 0.0175 abs/min. The chloroplast + dark group maintained a very small slope, and no chloroplast had a slightly larger slope. Figure two displays an absorbance versus time graph for spinach chloroplast reactions for all experimental groups. The chloroplast + dark group appears to have the highest absorbance at the end of 30 minutes, while no chloroplast has the lowest. Chloroplast + light appear to have a negative slope, at -0.0073 abs/min.

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