BISC220/S13: Mod 3 Lab 9

Lab 8: Cell Culture
Lab 10: Apoptosis - Protein 1
Lab 11: Apoptosis - Protein 2
Lab 12: Imaging Presentations
Media Recipes

Apoptosis

Programmed cell death is a mechanism by which cells kill themselves in a regulated way. Studies in the nematode worm C. elegans (all you 219 students should have fond memories of this little worm!) led to the discovery of the first genes regulating programmed cell death. Scientists wondered where 131 cells present in the last larval stage went as they were not present in the adult. These investigations led to the 2002 Nobel Prize in Medicine—the first of 3 for C. elegans!

Apoptosis is also required in higher eukaryotes for proper development. Without programmed cell death we would all have webbed fingers and toes! Some people with a defect in this pathway still do! Apoptosis also regulates some aspects of metamorphosis. We are all familiar with the tadpole "losing" its tail to change into a frog.

Apoptosis occurs in our bodies every day. The reason our tissues and organs do not grow out of control is the delicate balance between apoptosis and cell division. When one or both of these processes are not under tight control cancer can develop.

Apoptosis is the most common form of programmed cell death (PCD), a process by which a cell dies by undergoing a series of well studied morphological changes. The cells characteristically shrink, nuclear material condenses and fragments, the cytoskeleton collapses, and internal membranes disassemble. The cells also change shape, "blebbing" off membrane-bound parts. These cells are rapidly recognized by macrophages, engulfed and eliminated. As a result, this type of cell death does not elicit an inflammatory response. Another form of cell death called necrosis occurs as a result of cell trauma whereby cells burst and spill their contents out onto neighboring cells. This results in an inflammatory response that may be damaging to neighboring cells.

The system we'll be using to study the process of apoptosis in this three-part lab series is the HL-60 cell line, which is a line of partially differentiated cells in the granulocyte blood cell lineage that was derived from a patient with acute promyelocytic leukemia. In contrast to the 3T3 fibroblast cells you used last week, which formed an attached monolayer on the bottom of a tissue culture flask, HL-60 are suspension cells that grow unattached to any surface. (This should make sense to you based on their blood cell lineage.) HL-60 cells are particularly sensitive to apoptotic stimuli, so they are commonly used to study the events of apoptosis. The particular apoptotic stimulus we'll use is a drug called etoposide (also known as VP-16), which inhibits the enzyme topoisomerase II, an essential player in DNA replication and repair. Cells undergo apoptosis as a response to widespread DNA damage. You'll be using cells that have been treated with etoposide for different periods of time. In today's lab, you'll observe the time-dependence of one feature of apoptosis, DNA fragmentation. Although other agencies consider HL-60 cells to be a Biosafety Level 1 (BL1) cell line, Wellesley College has designated them to be BL2. Therefore, we cannot allow you to work with living cells in a course lab. You will instead be given etoposide-treated, fixed cells from which you will extract the DNA and analyze its size distribution by agarose gel electrophoresis.

Genomic DNA Isolation Protocol

Cells were treated with either 500 uM etoposide (VP-16) dissolved in dimethyl sulfoxide (DMSO) or DMSO alone as a control over a time course of 8 hours. Samples were taken at 2-hour intervals (0, 2, 4, 6 and 8). At each interval 1x10⁶ cells were pelleted in a microcentrifuge.

Before lab, 1 x 106 HL-60 cells were washed with 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 2 mM KH₂PO₄, pH 7.2) and pelleted.

To do in lab in lab today:

1. Resuspend all of your cells in 200 μL of PBS (phosphate-buffered saline) by vortexing at the highest speed. You may need to use a micropipet tip to dislodge some of the cells from the tube wall. Make sure that all cells are completely resuspended before proceeding to the next step.
2. Add 20 μL of proteinase K to the microfuge tube. Proteinase K is an enzyme which lyses (breaks open) cells and breaks down proteins. (Some of the proteins broken down by proteinase K include enzymes which might otherwise break down your DNA.)
3. Add 4 ul of RNase A (100 mg/ml), mix by vortexing, and incubate at room temperature for 2 minutes.
4. Add 200 μL Buffer AL and immediately mix by vortexing for 15 seconds. Make sure the lid on your tube is tightly closed before vortexing. This buffer contains a high salt concentration, which will help your DNA bind to the silica-gel-membrane of the spin column (below).
5. Incubate your tube at 56C for 10 minutes. This incubation gives the cells time to lyse.
6. Add 200 μL of 100% ethanol to the microfuge tube. Mix thoroughly by vortexing for 15 seconds.
7. Locate a DNeasy spin column with collection tube. Apply your sample to the DNeasy spin column, which is located in the collection tube. Microfuge at 8000 rpm for 1 minute. The DNA from the cells is now binding to the silica-gel-membrane in the spin column.
8. Remove the spin column and discard the flow-through by dumping the contents of the collection tube into your liquid waste container.
9. Remove the spin column and discard the flow-through into your liquid waster container; discard the collection tube (but NOT the spin column) into the autoclave bag. Place the spin column in a new, clean collection tube.
10. Open the spin column (which is sitting in its new collection tube) and add 500 μL of Buffer AW1. Microfuge at 8000 rpm for 1 minute.
11. Discard the collection tube and the flow-through. Place the column in a new collection tube.
12. Open the spin column and add 500 μL of Buffer AW2. Close the cap and microfuge at maximum speed (14,000 rpm) for 3 minutes. When removing your tube from the microfuge, be careful not to let the bottom of the spin column come into contact with the flow-through.
13. Remove the spin column from the collection tube and discard the collection tube.
14. Place the spin column in a labeled microfuge tube. (Label this tube on the side.) Use scissors to cut off the top of the labeled microfuge tube. Save this top.
15. Add 100 μL Buffer AE to the spin column. Be sure that you deliver Buffer AE to the membrane of the spin column but take care and don't touch the membrane with your pipet tip. Let the column sit at room temperature for 1 minute. Then, microfuge for 1 minute at 8000 rpm. The DNA will be eluted (pulled off) the membrane and be in the microfuge tube. Cap the tube with the top you had previously cut off. Buffer AE has a very low salt concentration, which helps pull the DNA off the silica-gel-membrane of the spin column.

Agarose Gel Electrophoresis Protocol

Prepare a sample for electrophoresis by mixing 10 μL of your genomic DNA with 2 μL of loading dye on a 1.0% agarose gel with SyberSafe stain. Run the gel at 100V for 45 minutes. The gel will be photographed using UV light and the photo posted to Sakai.

Template for loading the gel
Lane 1: NEB 1 kb ladder
Lane 2: Control 0 hours
Lane 3: Experimental 0 hours
Lane 4: Control 2 hours
Lane 5: Experimental 2 hours
Lane 6: Control 4 hours
Lane 7: Experimental 4 hours
Lane 8: Control 6 hours
Lane 9: Experimental 6 hours
Lane 10: Control 8 hours
Lane 11: Experimental 8 hours
Lane 12: NEB 100 bp ladder

What size is genomic DNA? What does it look like on the gel? What happens to the genomic DNA over time?