Major diseases Homeostasis and endocrine disorders

Laboratory experiment Techniques in cell death detection This laboratory experiment will demonstrate how apoptosis and necrosis can be measured using two different techniques, Flow Cytometry and Fluorescence Microscopy. Apoptosis is a regulated process of cell death where unwanted cells are eliminated where as necrosis is an accidental cell death considered being pathological. Although most cases of apoptosis are considered to be non-pathological there are several instances where either increased or decreased apoptosis is connected to human diseases. In some cases this might be due to the fact that the apoptosis has been incorrectly regulated and instead ended in necrosis. For this reason it is important to be able to distinguish between apoptosis and necrosis. Both type 1 and type 2 diabetes are characterized by a progressive beta-cell failure due to, mostly, apoptosis. Preventing this beta-cell death would help patients of both type 1 and type 2 diabetes to stay normoglycemic without exogenic insulin therapy. Although the two diseases share many similarities the cause of cell death seems to be fundamentally different in each case. The mechanisms behind beta-cell death and possible methods to prevent this are currently being investigated. Therefore it is important to be able to both distinguish between apoptosis and necrosis as well as measure these processes. In this laboratory experiment we will work with beta-TC6 cells (a mouse insulinoma cell line) that have been seeded in 35mm dishes. In addition to the control we will use heat shock and nicotinamide to induce apoptosis/necrosis. Apoptosis/necrosis will be detected by staining the cells with two fluorescent molecules, propidium iodide and bisbenzimide (Hoechst), which both binds to nucleic acids and thus can be used as nuclear markers. Propidium iodide, being unable to penetrate the cell membrane, is also used to detect cell membrane damage. In addition to this rather straightforward method we will also test a commercial kit that utilizes Annexin V to detect translocated phosphatidyl serine (PS). The flip of PS from the inner to the outer leaflet of the plasma membrane is a marker for early apoptosis. You can read more about the method in the attached documentation but please note that we have optimized the protocol given there. Before and after the laboratory experiment, each person should hand in answers to the questions at the end of this compendium. The answers should be short and concise and not total more then one page. E-mail your answers to Maria, [email protected]

 

Major diseases Homeostasis and endocrine disorders

Instructions The assistants will have added nicotinamide the day before and will place the dishes intended for heat shock in a 55°C water bath at the start of the laboratory experiment. Note that all centrifugations are done on the table top centrifuge for 10s at 2000 rpm. 1) When you receive your three dishes add the propidium iodide/bisbenzimide solution (1000X stock) to all of them and the assistants will return them to the incubator. 2) Incubate for 10 min. 3) Remove media from each dish and transfer to a separate eppendorf tube (this will contain floating cells) 4) Add 500µl trypsin and incubate for app. 5 min in the incubator (The assistants will tell you when the trypsination is done). 5) Centrifuge the tubes and discard the supernatant. 6) When the trypsination is done, add 500µl fresh media to the dishes to inactivate the trypsin. 7) Rinse the bottom of the plates to remove the cells and transfer everything to the eppendorf tubes. 8) Re-centrifuge the cells and remove the supernatant. 9) Resuspend the cells in 1ml cold media. (App. 1*106 cells/ml) 10) Transfer 100µl of the cell suspension from each experimental group into two new eppendorf tubes. One set of tubes will be used for FACS with only PI/Hoechst staining and one set will be stained using the Annexin V kit. The remaining 800µl are saved for microscopy. 11) Select the three tubes that should be stained with Annexin V and re-centrifuge the cells, discard the supernatant and resuspend cells in 1X Annexin V buffer. 12) Add 5µl of Annexin V (component A) to each tube. 13) Incubate for 15 min at RT. 14) During the incubation time the remaining 800µl can be re-centrifuged and resuspended in 20µl of fresh media. Add an additional 400µl media to the tubes containing cells only stained with PI/Hoechst and keep the cells on ice.

 

Major diseases Homeostasis and endocrine disorders

15) After incubation 400µl 1X binding buffer is added to the Annexin V stained cells. 16) Your group will start with either FACS or microscopy. FACS preparation: label six FACS tubes and transfer your cells to them. Start by analyzing the cells only stained in PI and then compare the result from the Annexin V staining. Microscopy preparation: 10µl is placed on slide and mounted with coverslip. Label the slides with your three experimental groups.

 

Major diseases Homeostasis and endocrine disorders

Pre-lab assignment Based on the previous experiences in this lab, students commonly do only what the lab assistants ask them to do, by adding materials one after another without thinking much about it. To be able to follow all steps during the lab and most importantly know what you are supposed to do, we want you to draw a schematic picture of all steps in the instruction in a way so that you can follow and understand it, without checking the protocol every time. (See the example)

Write your name and group number (1/4 – 4/4) on the paper, and then hand in your drawing by any of the following alternatives: 1. Scan your drawing in a KORINT-connected copy machine in BMC. The document will be saved as a pdf-file and sent to your @student.uu.se e-mail-address. Then send this to: [email protected] 2. In good light, take a picture of your drawing, with at least a 2 MP camera. Attach a high resolution copy of the picture to an e-mail, and send this to: [email protected] 3. You may also hand in your drawing in A2:103d. Put the paper in the box, labeled “Major Diseases”, on the chair outside the door to A2:103d.

Hand in your Pre-lab assignment no later than two days before your scheduled lab.

 

Major diseases Homeostasis and endocrine disorders

After-lab assignment: Questions 1) What are the characteristics of apoptosis? 2) Why is it possible to distinguish between apoptosis and necrosis by nuclear staining? 3) Explain the theory behind the Annexin V staining. 4) What are the advantages/disadvantages of using flow cytometry compared to fluorescence microscopy? 5) Describe the cell-signaling pathway induced by the nicotinamide and how it leads to apoptosis. 6) Explain the cause of beta-cell death in type 1 diabetes. 7) What are the major effects of beta-cell death on the body (i.e. the shift from normoglycemic to hyperglycemic)?

Submit your report in the Student Portal under “Assignments”, “Lab  report  2:  Answers  to  Questions  Flow  Cytometry”