Lab 2 Membranes W24 Final

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Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 29 Laboratory 2 Membranes Learning Objectives 1. Understand the role of plasma membrane in maintaining cell viability and integrity. 2. Test cell viability and permeability. 3. Understand that specific compounds can disrupt membrane integrity and cell viability. 4. Capture digital images 5. Employ software (ImageJ) to analyse image data. 6. Employ numerical analysis software to obtain statistical relevant information and build graphs representing data ( e g. Excel, Prism) 7. Employ multimedia software to generate a figure comparing data ( eg. Powerpoint, Adobe Illustrator). 8. Strengthen micropipetting techniques Experimental Objective 1. Build on your micropipetting technique 2. Identify compounds that can damage the cell membrane by scoring Trypan Blue exclusion and cell lysis. Laboratory Exercises Exercise 2.1: Membrane integrity and permeability NOTES : 1. Flowchart needed for lab 2. 2. All experiments are to be done in pairs. 3. All observations, drawings, and calculations are to be recorded in your lab notebook and must be completed during lab period. Your TA will need to sign your notebook at the end of the day. You will NOT receive marks if this is not done. 4. You will need to acquire image data and import into an approved digital device for future analysis (USB, laptop, phone, etc). You may need to upload these images as proof that you acquired them. 5. Each person is required to hand in their own report and data. 6. Before leaving, your lab book notes must be initialed by your lab partner and TA.

Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 30 **************************************************************************** Your lab notebook In this laboratory series, you are required to use a laboratory notebook to annotate your observations and answer questions. You may use a partitioned lab notebook for BLG411 as long as it is clearly labelled which section belongs to BGL411. A few comments about the notebook: 1. You should number your pages, starting with page 1 for the beginning of the partition dedicated to BLG411. 2. For each experimental lab (Lab 1, Lab 2, etc), you need to give: a. A lab title (as above). b. Complete date of your lab. c. Use subheading like 1.1, 1.2, etc. with a title for each exercise. d. Answer the relevant questions and make the relevant observations within the relevant subheading. e. At the end of the day, your TA will need to review and initial your lab book at the last page of your annotations for each specific day. f. If there are any calculations required for the lab, please complete them ahead of time in the flowchart and/or lab notebook. 3. You must not lose the lab notebook. Introduction to biological membranes (refer back to Chapter 10 and 11 of your book, if necessary) The membrane bilayer is fundamental to the very existence of life. The plasma membrane is the border between the non-living world and the cellular, living world. It acts as a barrier, preventing the cellular environment from homogenizing with the extracellular medium. A lipid bilayer lacking embedded proteins is impermeable to ions, polar organic molecules, proteins, carbohydrates, etc. However, it is semi-permeable to non-polar molecules, like O 2 , hydrophobic molecules, steroid hormones, and even small, polar molecules like water. A cell membrane cannot be like a wall because cells need to take up nutrients and expel waste. Thus, the cell membrane must allow for selective crossing of many types of molecules like ions, sugars, amino acids and so forth. This is often done by protein channels or transporters that are embedded within the membrane of the cell. Movement may follow a gradient, requiring no energy or it may be against a gradient, which requires energy. In eukaryotic cells, membranes are required to form organelles that each perform specific functions. For example, the endoplasmic reticulum can comprise over half of the surface area of the total membrane in a cell. Such an extensive surface area is needed as

Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 31 it is a synthesis hub for lipids, transmembrane proteins, secreted proteins, and proteins found in the lumen of organelles. In comparison, lysosomes are much smaller and take up less membrane area and volume. In many ways, lysosomes are specialized “garbage disposal” sites, where proteins and lipids are sent for degradation and turnover. Lysosomes are very rich in lipases and proteases that chew away lipids and proteins and exhibit low pH. Clearly, the membrane of the lysosome must be specialized to resist lysis, or otherwise, it would create havoc in the cell with the release of lytic enzymes. Studying biological membranes While membranes can be studied within living cells, the study of membrane composition requires isolation and subfractionation of the membrane components. Once isolated, the membranes can be solubilized using detergents and analyzed for proteins, lipids and carbohydrates. The study of biomembranes can be done with techniques requiring minimal equipment, such as osmotic shrinking or swelling of erythrocytes. Some techniques can be so complex and require a complete organic and physical chemistry lab. Often the chemical composition is of interest by itself, but more intriguing are the dynamic characteristics of membranes (such as semi-permeability) and functions (such as active transport and facilitated transport). To study these properties, researchers may synthesize artificial membranes in the form of lipid bilayers (also referred to as bilayer lipid membranes). The lipid bilayers are composed of natural or synthetic lipids that are artificially held between two aqueous environments. Artificial membrane research was significantly advanced by the addition of membrane components extracted from biological systems. The components have come from brain extracts, chloroplast or mitochondrial membranes, and have been augmented with oxidized cholesterol and any number of surfactants, which help solubilize or stabilize membranes. Researchers have been able to create fairly complex artificial membranes that can mimic nearly all of the qualities of a cell membrane. They have formed the lipid membranes as sheets spread across tiny apertures, or as small droplets within an aqueous environment. Sheets are more relevant to membrane permeability studies, while droplets (known as liposomes) are useful for analyzing cell fusion and membrane flow. Yet another approach to membrane analysis has been the use of markers or probes. The work utilizes either enzymes or fluorescent reagents. Fluorescent dyes have been covalently attached to specific membrane proteins, which can in turn be micro-injected into cells and their paths through the cell are monitored by computer aided video systems. With this new advancement, quantitative and qualitative analysis of membrane flow in a dynamic living cell is possible. There are also hydrophobic/amphipathic fluorescent dyes that intercalate into the lipid bilayer, thus fluorescently labeling membranes. Through all these approaches, a coherent theory for membrane structure has emerged. Most membrane analysts believe that essentially membranes are lipids in fluid suspension between two aqueous phases (inside and outside the cell), while the proteins are then attached to this lipid bilayer. However, lipids also interact with proteins through

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Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 32 hydrophobic or hydrophilic interactions, which may aid in giving membrane structure and form. Testing for plasma membrane integrity In this lab, you will perform one key experiment that relates to membrane semi-permeability and its role in maintaining cell integrity and viability. A central function of any membrane is to create a selective, semi-permeable barrier. If membranes were completely permeable or impermeable to all substances, life would not be possible. Studying conditions that disrupt membrane integrity is of course important. There are numerous treatments and events that can disrupt membrane function. Often these cause pores to form or extract lipids from the membrane like cholesterol. For example, a functional plasma membrane will normally exclude polar, bulky dye molecules like Trypan blue. However, if the plasma membrane is damaged, then Trypan blue can enter the cell to stain it. If sufficient damage occurs, cells will lyse and be lost. In this lab, you will investigate the effect of specific agents and deduct whether they damage the plasma membrane leading to loss of cell integrity and viability by quantifying Trypan blue exclusion from living cells.

Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 33 EXERCISE 2.1 – MEMBRANE INTEGRITY AND CELL VIABILITY KEY OBJECTIVE : Using Trypan blue exclusion, learn how to test if cells maintain membrane integrity, and thus viability, under different types of stresses. SCENARIO: As a recent graduate in biological sciences, you were hired by a contract company called VivioBio Ltd. that is known for its high-quality tests for cell viability and integrity. VivioBio received a large contract worth $350,000 for a 12-month project from a pharmaceutical company called Amigen. They identified 300 high-quality drugs using their artificial intelligence drug screening pipeline that are predicted to disrupt gram-positive bacterial cell walls. Such drugs have the potential to become new antibiotics. However, before proceeding further, Amigen needs to screen these drugs to find out which ones do not also damage mammalian cells. This is the contract, and what you will be doing at VivioBio. The project is rather complex with many assays. You will start with a simple assay before proceeding to the more complex high-throughput methods based on Trypan blue exclusion assay. FOR YOUR LAB REPORT : State your hypothesis: Compounds that cause plasma membrane damage will lead to loss of cell integrity and/or lysis. Questions to consider while you do the experiment : 1. Why is Trypan blue excluded from intact cells? 2. If Trypan blue stains cells, what does Trypan blue bind to within cells? 3. What is Tween-20? Quick Questions – answer in your lab book: . 1. You are given a stock solution of 10% Tween-20 in PBS. Describe the process to make 2 mL of 1%, 0.5% and 0.05% Tween-20 in PBS. Materials • 5-coverslips with J774 macrophage cells in a 6-well plate • Trypan Blue stain • 10% Tween in PBS • 1x phosphate-buffered saline (PBS) • Microcentrifuge tube • Hepes-buffered RPMI (HPMI) • Drug G:________________ • Drug K: ________________ • Mounting media

Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 34 • Slides and mounting media • Microscope • Pipetteman (P10, P100, P1000) • White, yellow and blue pipette tips • Phone adaptor • Camera device (smartphone) Procedure 1. Obtain the microscope from the designated cupboard and set it up as in Lab 1. 2. Mount the camera adaptor and your camera device as before. EXPERIMENTAL SET UP NOTE: Use the same phone through this experiment A. You will have five-samples set up in a 6-well plate (Fig 2.1). I. One will be cells incubated with HPMI media. This is expected to have no effect on your cells. It is therefore a negative control , which will be important for you to define “normal” or “no effect” condition. II. Then, you will have two concentrations of Tween-20: 0.5% and 0.1%. These should be positive controls . Tween-20 is a detergent that if at high enough concentration, should lead to extensive cell damage and even lysis. So, it can be a positive control for cell loss and is useful to show that your experimental setup works. III. Normally, you would also include a “solvent” or “ vehicle” control . This is a control that aims to identify any effect that the solvent/carrier of the test drugs may have on your assay. However, to simplify the experimental set-up we will skip this control for this lab. IV. Overall, remember that good experimental design includes both negative controls as a benchmark of what “no effect” looks like and a positive control to demonstrate that your experimental design works and is not flawed. 3. Using the P1000, add 1 mL of HPMI into five microcentrifuge tubes and label each as HPMI, 0.5% Tween-20, 0.1% Tween-20, drug G and drug K. 4. Using the 10% Tween stock solution, add enough to the Tween tubes to make 0.5 and 0.1% Tween-20. Make sure to use the appropriate micropipette. Show this calculation in your lab notebook. SHOW THE CALCULATION HERE AND IN YOUR REPORT: 5. For drugs G and K add 20 μL to the corresponding tubes. 6. Take one 6-well plate with macrophages plated on coverslips (there should be five coverslips) from the incubator. Label with your group name. 7. Aspirate the media by placing the tip of the aspirator in the corner of the well.

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Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 35 8. Using the P1000, pipette 1 ml of 1x PBS to wash cells by gently dispensing the PBS to the side of the well and gently swirling the 6-well plate. 9. Aspirate PBS. 10. Pipette 1 ml of each condition into the corresponding well of the 6-well plate. Figure 2.1 Set up of 6-well plates. 11. Incubate cells at 37  C for 20 minutes. 12. After the incubation, aspirate liquid from wells and add 1 ml of Trypan blue to the cells and incubate for 7 min. 13. Aspirate liquid from wells and add 1 ml of PBS to wash cells. NOTE: Dispense the PBS gently to the side of the well, NOT on top of the cells. 14. Repeat washing step 14, twice. 15. Aspirate the last PBS wash. 16. Add fresh 1 mL PBS into each well after washing. NOTE: Dispense the PBS gently to the side of the well, NOT on top of the cells The trypan blue stain should be all gone. 17. Add a drop of mounting media to a slide. 18. Pick up a coverslip (don’t drop!) and place the coverslip edge gently on to a Kimwipe to remove excess liquid. Mount the coverslip on a slide – make sure cells are facing down onto the slide (i.e. invert the coverslip then mount on the slide) 19. Put slide on microscope, coverslip facing the 4x objective. Focus the slide at 4x. Rotate nosepiece to 10x and focus before moving on to the 40x objective. 20. At 40x magnification, acquire at least FIVE images of at least FIVE different random fields of view, using your phone and the phone adapter for the microscope. NOTE: You will need these images to quantify the total number of cells in each field and the percent of these cells that are stained with Trypan for your report using these images. HPMI 0.5% Tween Drug G 0.1% Tween Drug K

Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 36 21. When finished acquiring images, repeat for a different coverslip until you examine all conditions. After completing this laboratory, you should be able to define or explain the following terms: Membrane Membrane integrity Lipid extraction Permeability Detergent Cell viability assays Membrane semi-permeability Cell viability microliter Micropipetting Micropipettes

Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 37 LABORATORY REPORT 2: Membranes (Total = 51 marks) Aim of the laboratory report: To learn how to use image/microscopy data to test hypotheses. Quantitatively and qualitatively assess image/microscopy data Generate informative data graphs that help make and support your conclusions Generate informative figures that help make and support your conclusions Tools at your disposal: Your data ImageJ software for image handling and analysis Spreadsheet like Excel Multimedia software like Powerpoint Library Internet You may use the library and/or online tools to find answers to various questions for which the answer may not be in the lab manual, lecture or book. When you do this, you must follow this procedure for EACH question that you had to research the answer: For each research question, in your answer you MUST INCLUDE: o The search engine used (Google, Pubmed, Google scholar, etc). I strongly recommend you use www.pubmed.com or scholar.google.com to find research articles and reviews. o The terms used in your search o Original references (your search engine and the link within it are usually not acceptable as a reference. A Google image or statement in Wikipedia is not a reference since they did not produce the original observation. You should reference the original paper or source. Alternative references may include academic and commercial (biotech/pharma) websites. NOTE: Failure to follow the above procedures will result in a severe penalty even if you got the correct answer (this is not negotiable). COVER PAGE (- 2 if missing or incomplete) INTRODUCTION : Not required for this lab report. RESULTS (41 marks – includes Hypothesis, Part A and B)

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Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 38 EXERCISE 2.2 – MEMBRANE INTEGRITY AND PERMEABILITY Hypothesis: Drugs that damage the plasma membrane will disrupt cell integrity causing cell loss or staining with Trypan blue. Include this hypothesis in your report. /1 Part A: Data Analysis and Results ( /26 ) 1. We have created a spreadsheet template for you to use in this experiment. Please open Lab2_Membrane_Damage.xls in Lab Appendix and Templates in D2L. 2. Using ImageJ, open the 5 images that you acquired for cells exposed to HPMI. Again, this condition serves as your negative control in that you should expect cells to look “normal”. 3. Count the total number of cells in each image. Then, count the number of cells that were trypan positive. Enter this information in the spreadsheet template. Make sure the total and trypan positive number in each row come from the same image. 4. The template is already set up to calculate the average number of cells per field for each condition and the percent of cells that are trypan positive for each condition. These values should appear automatically as you enter your image data. NOTE: While this is given to you in this lab, you should review Excel Appendix and learn about: Cell merging, centering text, functions for average and percent, and organizing tools to make it easy to organize and observe your data. 5. Repeat this process for each experimental condition. a) FOR YOUR REPORT: Print the tables for your data as shown and include in your report . /2 6. Build a column graph that represents the total cells per image vs. treatment. Your column graph should have a title, y-axis and x-axis titles, and each bar needs to have a label. You must add standard deviation to each bar. See Appendix on Excel on how to do this. Save this graph as an image called cell number as a .jpg or .png. a) FOR YOUR REPORT: To decide on whether any treatment causes a significant change in a given property, you would normally repeat this experiment at least three independent times (on different days), then find the mean/median, and then apply a statistical test. However, we do not have time to do this. So, we are going to assume that each image is an independent data point for the purpose of running a statistical test and you will ask if your drugs are statistically different from the positive and negative controls to determine if a drug has no effect or is detrimental to cells. First, just by looking at your data trends, state your opinion on whether Drug G or Drug K damage mammalian cells. Explain your answer. /6

Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 39 7. Since you have five samples, you should not use Student’s t -test, which is normally used to test statistical difference between two conditions only. In most cases, you should not run a series of t-test to compare pairwise conditions when your experiment has more than two conditions. You should use ANOVA and a post- hoc test (Tukey’s test). Recognizing that you may not have learned about this yet, you will only be expected to use an online calculator to make your conclusions. 8. Go to http://vassarstats.net/anova1u.html . • Enter “number of independent samples” = 5 • Click on independent samples and unweighted. • You will run this test twice. • First, you will enter the total number of cells per image. Column 1 will be on HPMI (copy and paste total cells from your Excel file), repeat for the other samples. There should be five values for each treatment, where each value is the number of cells per image. • Press calculate and magic will happen. a) FOR YOUR REPORT: Look for ANOVA summary and p value. Write this down in your report. If p<0.05, the conditions in your experiment are statistically different. You just don’t know in what way your data is different. b) Now look at Tukey’s test. This assesses each condition pairwise (for example, column 1 with column 2 (M1 vs M2), column 1 with column 3 (M1 vs. M3), etc. Do a print screen and include this in your report. This is your key result to be discussed below. /6 c) Repeat this process starting at instruction H above for percent positive cells. /6 9. Build a graph that shows the average total cells per image and the average percent trypan-positive cells vs. treatment. Your column graph should have a title, y-axis and x-axis titles, each column needs to have a label. You must add standard deviation to each bar. See Appendix on Excel on how to do this. Save this graph as an image called percent trypan positive as a .jpg or .png. b) FOR YOUR REPORT: Now using the ANOVA and Tukey’s test results for both total number of cells and percent Trypan-positive data, state your conclusion on whether Drug G or Drug K damage mammalian cells or not. Explain your answer by using and comparing your data for cell number/image and the ANOVA tests. /6

Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 40 Part B: Representing your data ( /14 ) 1. To make a figure with your images and column graph, open Powerpoint and make a new slide. Use Page Setup to select “Letter” size and upright orientation. 2. Go to ImageJ and open an image that depicts a representative result for the HPMI, 0.1% and 0.5% Tween-20, and Drug G and Drug K conditions. Using the “Rectangular tool”, select 1/3 of the area of one image. Click “Edit” and then “Copy to the system” (don’t just copy) and paste into Powerpoint. 3. Back in ImageJ, click on “Analyze” and select Tools. Look for ROI (stands for regions of interest) manager. Make sure the first image that you used to create the rectangular box is active (meaning last image you touched with the cursor), click on “ Add ” in the ROI Manager. This will cause a code that represents this ROI to appear in the ROI manager. 4. Now click on the other images and then select the ROI code. The box should now appear in the other images, which you can move around independently to 1/3 of the area of each image. All images must be the same size in Powerpoint without stretching or condensing the scales. Copy into the system as before. Paste into Powerpoint. a) Figure 1: Generate a figure in Powerpoint to include in your report. Figure 1 should show one image for each condition. Images should be aligned and same size. Label with the condition and add scale bar. Include a descriptive figure legend with title. /6 b) Figure 2: Generate a figure in Powerpoint to include in your report with two parts. A) Is the column graph showing average cell number per field. B) Is the column graph showing average percent trypan-positive cells. Include a descriptive figure legend with title. /6 5. Overall, write a conclusion about the effect of drug G and K on cell viability based on the statistical data and by comparing to the negative and positive controls. /2

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Based on W.H. Heidcamp Edited by C. Antonescu R. Botelho and L. Victorio BLG 411 Membrane Integrity and Permeability Experiment designed by R.B. and C.A. Cell Biology Lab 41 Part C: Research question: /10 C1. Filipin is a fluorescent molecule that can bind cholesterol. At lower concentrations, filipin can be used to label membrane and quantify cholesterol based on fluorescence intensity. At higher concentrations, it may extract cholesterol from bilayers, and/or occlude cholesterol, and/or aggregate cholesterol, then allowing one to study the functions of cholesterol. Using pubmed.com, search for “cholesterol filipin” AND Year according to the following rule (if your last name starts with an A, include 1997 in your search, the letter B, 1998, letter C, 1999, etc. letter Z, 2022). Then scan through the list and pick a research paper (do NOT use a review article) where the authors used filipin – don’t be afraid to look through a bunch and don’t be boring and pick the first article you see!! a. In your own words, explain what they used filipin for in this paper. /2 b. What did they observe in using filipin? Make sure to refer to relevant figures and describe this to support your statements. /4 c. What conclusion to they make by using filipin? /2 d. Attach the pdf of the paper to your report. /2

Lab 2 Membranes W24 Final

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