8.10 Experimental Techniques

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Studying Apoptosis

 

As the chapter has discussed so far, apoptosis is the ablation of cells controlled genetically during normal development. The destructive process is characterized by biochemical and morphological changes that occur in the cell; such as, change in mitochondrial membrane potential, DNA fragmentation, membrane blebbing, caspases activation, and formation of apoptotic bodies (1). To study apoptosis in individual cells, a number of methods can be applied based on the changes discussed in the aforementioned. In this section, the assays predominantly used for studying apoptosis individual cells are based on: DNA fragmentation, chromatin morphology, DNA strand breaks, phosphatidylserine markers, and plasma membrane alterations of permeability (1).

 

DNA Fragmentations

 

DNA strand breaks occur when nuclear enzymes are activated, resulting in the fragmentation of DNA chromatin structure. This biochemical hallmark of apoptotic cells is characterized by the presence of DNA fragments at multiples of 180-200 bp, which are separated in a ladder-like trend similar to a marker ladder in agarose gel electrophoresis (6). While detection of apoptosis-induced DNA fragmentation using gel electrophoresis offers a way to visually inspect the level of fragmentation seen in a cell and itspopulation, the method does not provide crucial infomration regarding the localization of DNA fragmentations (2). To measure the activity of DNA breakage in individual cells, labeling or staining of the cellular DNA is performed and subsequently analyzed by flow cytometer, fluorescence microscopy, or light microscopy.  The two common labeling techniques to observe DNA content in apoptotic cells are based on using either modified nucleotides (e.g., biotin-dUTP, fluorescein-dUTP) to label DNA, or fluorescent DNA-binding dyes (DNA fluorochromes) to stain DNA. Cellular DNA labeled with modified nucleotides is performed using exogenous enzymes that interact with the DNA, such as terminal transferase or DNA polymerase. On the other hand, DNA stained with DNA fluorochromes  is accomplished by intercalating the dyes into DNA, where they can be projected to become highly fluorescent. As a result, cells undergoing apoptosis contain less bound dye molecules since they lose DNA during the staining process (1).

 

TUNEL Assay

 

Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) is an assay used for localizing apoptotic DNA fragmentation. This methodology encompasses the use of labeled nucleotide, dUTP, as a means of identifying blunt ends of double-stranded DNA breaks caused during apoptosis. During this time, TdT performs the addition of labeled dUTP to the 3’-hydroxyl termini of DNA ends, which can then be visualized by immunohistochemical techniques (2). The types of available modified nucleotide include fluorescently labeled dUTPs, biotinylated dUTPs, digoxigenylated dUTPs, and bromine-labeled dUTP (BrdUTP). Although TUNEL assay offers a method to visually inspect the localization of apoptotic DNA fragmentation in situ, its accuracy in detecting apoptosis must be re-confirmed using another independent method. This process is due to the fact that DNA damage occurs not only occurs due to apoptosis, but also necrosis (2).

 

Chromatin Condensation

 

Detection of apoptosis in cells can also be accomplished by detecting the level of condensed chromatin. During apoptosis, the nuclear chromatin undergoes a phase change whereby the genomic structure is turned into a condensed form, which is sequentially fragmented and packed into apoptotic bodies (8). Using nucleic acid stains, apoptotic and normal cells can be distinguished from a cell population by either flow cytometry or fluorescence microscopy. Some dye stains include YO-PRO-1, which generates a green fluorescence; and Hoechst 33342, which shows a blue fluorescence (8).

Gamma-linolenic acid (GLA) is determined to induce apoptosis in leukemia cells by staining the chromatin with Hoechst 33342 (9). Compared to the control group, GLA-treated cells undergone apoptosis and they are characterized by the presence of the tightly-packed chromatin (9). Specific quantification of apoptotic cells is done using flow cytometry coupled with the nucleic stains referred in the aforementioned (8). The cells are then subsequently analyzed by flow cytometry using dual excitations to distinguish between viable cells (V), necrotic cells (N), and apoptotic cells (A). C

DNA Strand Break and DNA Fragmentation Assays

 

Both of these techniques make use of DNA strand breakage during the later stages of apoptosis as their target. Strand breakage assay analyzes nick translation via exogenous DNA polymerase while DNA fragmentation assay measures end labelling via exogenous terminal transferase (3). The bases of both techniques are the incorporation of marked nucleotides that can be traced in some way (fluorescent dUTPs, fluorescent protein binding dUTPs, etc.), with the former fixing nicks and the latter adding dUTPs to ends (3).

Cells are grown with or without the apoptotic factor, and then fixed using formaldehyde (4). Their respective enzyme are added and allowed to incubate along with marked dUTPs. A secondary reagent step may be needed to label the dUTPs with something visible. The respective levels of strand breakage/DNA fragmentation can then be compared and related back to apoptosis.

Limitations of the assay include time, labour and specificity, with DNA fragmentation taking a shorter amount of time and having a higher sensitivity to apoptosis compared to other DNA breakage possibilities such as necrosis (4).

 

Phosphatidylserine Assays

 

This assay makes use of the phosphatidylserine protein, which, during apoptosis/necrosis, would translocate from the inner side of the plasma membrane to the cell surface (5). Labelled Annexin V, which binds to certain phospholipids normally found on the inner side of the membrane, makes a highly selective marker for this apoptotic change. Due to the loss of plasma membrane integrity during necrosis (no loss during apoptosis), a non-permeable dye such as propidium iodide (PI) would be able to differentiate between the two kinds of cell death. Make sure the label on Annexin V is of a different color as the dye used for necrosis determination.

Cells are grown with or without the apoptotic factor and then incubated with labelled Annexin V. A secondary reagent step may be needed to label Annexin V with something visible. The non-permeable dye, PI, would now be used to color the cells which are unable to exclude it (necrotic cells). Perform flow cytometry to visualize the amount of apoptotic cells (Annexin+/PI-), necrotic cells (Annexin+/PI+) and non-apoptotic/necrotic cells (Annexin-/PI-).

 

­Plasma Membrane Damage Assays

 

These are simple assays to determine the difference between living cells and dead cells by use of plasma membrane exclusion. Quick and inexpensive, these two assays offer a way to differentiate in both light microscopy and flow cytometry. Trypan Blue is a staining dye that binds to intracellular proteins while PI is a fluorescent dye that binds to DNA (4). The procedures are simply to incubate the cells with these dyes and read the results with either light microscopy (trypan blue) or flow cytometry (PI); with labelled cells being dead cells.

These assays are designed to be quick and inexpensive, and thus are not very specific towards apoptosis. Trypan Blue in particular only detects very late apoptotic cells. However, these assays also use a lot fewer cells than most other assays, making them efficient to use.

 

Studying Senescence

 

Senescence associated-β-galactosidase (SA-β-gal) activity

 

Cellular senescence can be induced by either exhaustion of replicative capacity or exposure to cellular stress. The markers that correspond to senescence thus far are cellular morphology, telomere length, and senescence associated β-galactosidase (SA-β-gal) activity (10). In senescent cells, lysosomal beta-galactosidase is accumulated over time, which can then used to measure and detect cells undergoing senescence. β-galactosidase are a collective of enzymes that cleave nonreducing β-D-galactose residues from glycoproteins, sphingolipids, and keratan sulfate in β-D-galactosides (10). For SA-β-gal assay, activity of SA-β-gal is detected using cytochemistry coupled with 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) as a substrate, where SA-β-Gal catalzyes the hydrolysis of X-gal, which then produces a blue color in senescent cells. During this time, fibroblasts are stained and SA-β-gal positive cells are counted using a fluoresence plate reader or flow cytometry (10).    


References

  1. Roche Diagnostics (2014). Apoptosis Assay Methods. Retrieved from http://www.roche-applied-science.com/shop/products/apoptosis-assay-methods
     
  2. Kyrylkova, K., Kyryachenko, S., Leid, M., & Kioussi C. (2012). Detection of apoptosis by TUNEL assay. Methods Molecular Biology, 887: 41-7. Doi: 10.1007/978-1-61779-860-3_5.
     
  3. Gold R., Schmied M., Rothe G., Zischler H., Breitschopf H., Wekerle H., & Lassmann H. (1993). Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections. J Histochem Cytochem, 41(7): 1023-30
     
  4. http://www.roche-applied-science.com/shop/products/apoptosis-assay-methods
     
  5. Vermes I., Haanen C., Steffens-Nakken H., & Reutelingsperger C. (1995). A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol Methods. 184(1): 39-51
     
  6. Cheah Y.H., Nordin, F.J., Sarip, R., Tee, T.T., Azimahtol, H.L., Sirat, H.M., Rashid, B.A., Abdullah, N.R. & Isamil, Z. (2009). Combined xanthorrhizol-curcumin exhibits synhergistic growth inhibitory activity via apoptosis induction in human breast cancer cells MDA-MB-231. Cancer Cell International, 2;9:1. doi: 10.1186/1475-2867-9-1.
     
  7. Kalghatgi, S. (2009). Non-thermal Plasma Initiated Apoptosis in Cancer Cells. Retrieved from http://www.sameerkalghatgi.com/Cancer%20Therapy.html
     
  8. Invitrogen. (2010). Chromatin Condensation/Membrane Permeability/Dead Cell Apoptosis Kit with Hoechst 33342/YO-PRO-1 and PI for Flow Cytometry. Retrieved from http://tools.lifetechnologies.com/content/sfs/manuals/mp23201.pdf
     
  9. Haitao, G., Kong, X., Shi, L., Hou, L., Liu, Z., & Li, P. (2009). Gamma-linolenic acid induces apoptosis and lipid peroxidation in human chronic myelogenous leukemia K562 cells. Cell Biology International, 33: 402-410. doi: 10.1016/j.cellbi.2009.01.014