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

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

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