Apoptosis and Measurement of Cell Death

German Scientist “Carl Vogt” was first to describe the principle of apoptosis in 1842. In 1885, Anatomist “Walther Flemming” gave more precise description of Programmed Cell Death. Apoptosis is a form of Programmed Cell Death that occurs in multicellular organisms. It is a Greek word which means falling off. It leads to breakdown and disposal of cells. Macrophages and other Phagocytic Cells remove them by Phagocytosis, without developing any type of inflammation. It is a biochemical event that leads to morphological changes and death. The average adult human looses 50-70 billion cells each day due to apoptosis.

1. RANI DURGAVATI VISHWAVIDHYALAYA JABALPUR (M.P) A Presentation On “Apoptosis And Measurement Of Cell Death” Presented By: Ambika Prajapati M. Sc. Biotechnology I Semester

2. Apoptosis Apoptosis is a form of Programmed Cell Death that occurs in multicellular organisms. It is a Greek word which means falling off. It leads to breakdown and disposal of cells. Macrophages and other Phagocytic Cells remove them by Phagocytosis, without developing any type of inflammation. It is a biochemical event that leads to morphological changes and death. The average adult human looses 50-70 billion cells each day due to apoptosis. German Scientist “Carl Vogt” was first to describe the principle of apoptosis in 1842. In 1885, Anatomist “Walther Flemming” gave more precise description of Programmed Cell Death.

3. Why do cells undergo apoptosis? Apoptosis is a general and convenient way to remove cells that should no longer be part of the organism. Some cells need to be “deleted” during development – for instance, to whittle an intricate structure like a hand out of a larger block of tissue. Some cells are abnormal and could hurt the rest of the organism if they survive, such as cells with viral infections or DNA damage. Cells in an adult organism may be eliminated to maintain balance – to make way for new cells or remove cells needed only for temporary tasks.

4. At the centre of the process of programmed cell death , is a family of proteases named “Caspases”, involved in the initiation and execution of Apoptosis. Examples are as follows : Caspases 9, Caspase 3, Caspases 8, Caspases 10. They can be activated by a large number of stimuli via two central pathways : One involving Mitochondria, Other using Trans-Membrane Receptors of the Tumor Necrosis factor (TNF). Intrinsic Pathway Extrinsic Pathway Pathways of Apoptosis

5. Intrinsic Pathway Activation of apoptosis via Mitochondria is an Intrinsic Pathway where stress signals, DNA damage signals and defects in signaling pathways are processed. The Intrinsic Pathway uses the “Mitochondria” as a central component for activation of apoptosis. In this pathway, various stresses lead to activation of the pro-apoptotic proteins Bax / Bak which induces release of Cytochrome c from Mitochondria, formation of the Apoptosome and activation of the initiator Caspase 9. Finally, the executioner Caspase 3 is activated and cells are destructed by Proteolysis. Apoptosis via this pathway can be controlled by various Anti-Apoptotic Proteins including the Bcl-2 and Bcl-X2. (Bad, Bim , PUMA, NOXA – helps in apoptosis.)

6. Extrinsic Pathway This pathway uses Extracellular Death Ligands ( Fas ligand, Tumor Necrosis Factor ) to activate “Death Receptors” which pass the apoptotic signal to initiator Caspase 8 and to the executioner Caspases 3 and 7. In the execution phase of apoptosis, various cellular substrates are degraded leading to cellular collapse. As a consequence of caspase activation, enzymes and protiens are degraded, leading to cell death. The stimuli that induce apoptosis include DNA damage, stress conditions and malfunctions of pathways regulating cell proliferation.

7. Measurement of apoptosis The selection of detection assays is based on morphological criteria and distinguishable marks of apoptotic pathways. The detection of apoptosis includes methods like membrane alterations, DNA fragmentation, cytotoxicity, cell proliferation, mitochondrial damage, immunological detection and mechanism based assays. There are many ways by which we can detect and measure the cell death (apoptosis). Here, I am going to discuss few of them: DNA Ladder Assay. Annexin V/ PI Assay. Tunel Assay.

8. DNA ladder assay DNA fragmentation is a hallmark of apoptosis in mammalian cells. “Abcam’s Apoptotic DNA Ladder Detection Kit” (generally used) provides an easy and sensitive means for detecting DNA fragmentation in apoptotic cells. During apoptosis, the DNA strand is cleaved by CAD and generates a number of DNA fragmentations of 180-200 base pairs known as DNA ladders. These DNA fragments can be extracted from the cells and visualized using agarose gel electrophoresis. Kit Components : TE Lysis Buffer 1.8 mL, Enzyme A Solution 0.25 mL, Enzyme B (Lyophilized) 1 vial, Ammonium Acetate Solution 0.25 mL, DNA Suspension Buffer 1.5 mL . Additional Materials Required : Microcentrifuge, Isopropanol, Pipettes and pipette tips, Orbital shaker, 70% ethanol, 1.2% agarose gel containing 0.5 μ g/ml ethidium bromide. * Store kit at -20°C.

9. Assay protocol 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. Pellet 5-10 x 10⁵ cells in a 1.5 ml microcentrifuge tube. For adherent cells, gently trypsinize cells and then pellet cells. 3. Wash cells with PBS (not provided) and pellet cells by centrifugation for 5 min at 500 x g. Carefully remove supernatant using pipette. 4. Lyse cells with 35 μ l TE Lysis Buffer, gentle pipetting. 5. Add 5 μ l Enzyme A Solution, mix by gentle vortex and incubate at 37°C for 10 min. Note: If cells contain high levels of DNase, then the incubation step should be skipped, as high level DNase can digest DNA ladder generating smear pattern. 6. Add 5 μl Enzyme B Solution into each sample and incubate at 50°C for 30 min or longer (overnight is OK).

10. 7. Add 5 μl Ammonium Acetate Solution to each sample and mix well. Add 50 μl isopropanol (not provided), mix well, and keep at -20°C for 10 minutes. 8. Centrifuge the sample for 10 minutes to precipitate DNA. 9. Remove supernatant, wash the DNA pellet with 0.5 ml 70% ethanol, remove trace ethanol, and air dry for 10 minutes at room temperature. 10.Dissolve the DNA pellet in 30 μl DNA Suspension Buffer. 11.Load 15-30 μl of the sample onto a 1.2% agarose gel containing 0.5 μg /ml ethidium bromide in both gel and running buffer. 12.Run the gel at 5 V/cm for 1-2 hours or until the yellow dye (included in the suspension buffer) run to the edge of the gel. 13.Ethidium bromide-stained DNA can be visualized by transillumination with UV light and photographed. ENZYME B SOLUTION: Dissolve Enzyme B with 275 μl H₂O and mix well before use. The Enzyme B solution should be frozen at -80°C immediately after each use, or aliquot and then stored at -80°C for future use.

11. PROTOCOL SUMMARY Induce apoptosis in cells Lyse the cells Add Enzyme A Solution Add Enzyme B Solution Dissolve DNA Pellets in Suspension Buffer Run on Agarose Gel Visualize by UV - Transilluminator DNA Ladder Assay [as viewed under UV - Transilluminator (Detection Result )]

12. Annexin V and pi staining assay This assay is used to detect mid to late stage cell death. An early event in apoptosis is the flipping of phosphatidylserine of the plasma membrane from the inside surface to the outside surface. Annexin V binds specifically to phosphatidylserine and labelled Annexin V can be used detect apoptotic cells.  Propidium Iodide is used in conjunction with labelled Annexin V. The cell membrane integrity excludes Propidium Iodide in viable and apoptotic cells, whereas necrotic cells are permeable to Propidium Iodide. The data generated by flow cytometry are plotted in two-dimensional dot plots in which PI is represented versus Annexin V. These plots can be divided in four regions corresponding to: 1) viable cells which are negative to both probes, 2) apoptotic cells which are PI negative and Annexin positive, 3) late apoptotic cells which are PI and Annexin positive, 4) necrotic cells which are PI positive and Annexin negative. Necrotic Cells Flow Cytometry Analysis Plot

13. Assay protocol Cell Preparation is done by adding 1x PBS and 1x Annexin V Binding Buffer. Incubate cell suspension tubes in the dark for 15 minutes at room temperature. Again add 100 μ L of 1 x Annexin V binding buffer to each reaction tube. There should be approximately 200 μ L in each tube. Add 4 μ L of PI that has been diluted 1:10 in 1 x Annexin V binding buffer (i.e. 1 μ L PI with 9 μ L 1 x Annexin V binding buffer). Again i ncubate the tubes in the dark for 15 minutes at room temperature. Add 500 μ L 1 x Annexin V binding buffer to wash the cells. Centrifuge samples at 335 x  g  for 10 minutes and decant the supernatant. Resuspend cells in 500 μ L 1 x Annexin V binding buffer and 500 μ L 2% formaldehyde to create a 1% formaldehyde (fixative) solution. Mix tubes by gentle flicking.

14. 9. Fix samples on ice for 10 minutes. 10. Add 1 mL 1 x PBS to each sample and mix gently by flicking. 11. Centrifuge tubes at 425 x  g  for eight minutes and decant the supernatant. 12. Repeat steps 10 and 11. 13. Resuspend pellet by flicking the tube. 14. Add 16 μl of 1:100 diluted RNase A to give a final concentration of 50 μg /mL. Incubate for 15 min at 37°C. 15. Add 1 mL 1 x PBS  and mix gently by flicking. 16. Centrifuge tubes at 425 x  g  for eight minutes. 17. Samples are now ready to be analyzed by Flow Cytometry.

15. PROTOCOL SUMMARY Harvest the cells Induce Apoptosis Add Annexin V and PI Stain Visualize under Flow Cytometer Annexin V and PI Staining Assay [as viewed under FC (Detection Result)]

16. Tunel assay TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, also called the TUNEL assay, detects the DNA breaks formed when DNA fragmentation occurs in the last phase of apoptosis. The TUNEL staining / TUNEL assay method relies on the enzyme terminal deoxynucleotide transferase (TdT), which attaches deoxynucleotides to the 3’-hydroxyl terminus of DNA breaks. The method is based on the ability of TdT to label blunt ends of double-stranded DNA breaks independent of a template. TUNEL assay is a sensitive and easy tool to identify late stage apoptotic cells and an useful approach to develop further insights in disease pathogenesis and physiological mechanisms. There are many labels that can be attached by TdT to the DNA fragments such as; Fluorescent-tagged dUTP - FITC TUNEL assay biotinylated dUTP DIG or digoxigenylated dUTP

17. Assay protocol SAMPLE PREPARATION:  TUNEL assay can be performed in any type of sample (tissue sections, cell suspensions, cell lines). It is essential to fix the sample with an appropriate fixative (1-4% paraformaldehyde) and permeabilized with either methanol or  Tween-20 . Samples can be stored at –20°C for further use or can proceed directly for the next step. TUNEL staining:  The staining requires positive and negative controls along with the test cell samples. All the samples undergo incubation with TdT enzyme mixture for a specific time interval followed by labeling with the antibody mixture. DNA fluorochromes such as BrdUTP and propidium iodide are used for background nucleus marking. ANALYSIS:  The results can be observed through flow cytometry (cell suspension) or light or fluorescent microscopy ( adhered cells or tissue sections) depending upon the type of labeling.

18. PROTOCOL SUMMARY Fix the sample with Fixative and permeabilized with Tween-20 Sample undergo incubation with TDT dUTP are labelled with Fluorochromes (BrdUTP) Visualize under Flow Cytometer Tunel Assay [as Viewed under FC (Detection Result)]

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