3220823

From CellBiology

Staining Necrotic Cells

Detection of cells undergoing a necrotic type of cell death works on the fact that, in contrast to programmed cell death such as apoptosis, the cells membrane becomes permeable in its very early stages. Since loss of membrane integrity is a distinguishing morphological feature of necrotic cell death, adapting techniques using membrane impermeant dyes such as propidium iodide (PI) and ethidium homodimer are preferred as small charged molecules that would not normally be able to cross the membrane are consequently able to do so. These are often used in conjunction with various other stains such as the Hoechst stain, trypan blue and TUNEL-stain, ultimately double or even triple staining to determine more specific criteria such as nuclear morphology and cellular morphology [1]. Such procedures are necessary as cells in the late stages of apoptotic cell death are also permeable to all of these dyes due to secondary necrosis.


Membrane permeability detection

The intact membranes of living cells prevent cationic dyes such as PI and trypan blue from entering, and these dyes are therefore used for membrane permeability detection[2] . Two of the main live/dead cell assays used in immunochemistry are; ethidium homodimer and PI as they exploit this feature of necrotic cells.

Figure 1. Calcein-AM and Ethidium Homodimer stain

Apoptosis/Necrosis Detection Kit 1

Apoptosis/Necrosis Detection Kit 2

Ethidium homodimer

The ethidium homodimer assay can be used to detect dead or dying cells. It is a membrane impermeable fluorescent dye which binds to the DNA[3] . When cells undergo necrosis, the plasmalemma of the cells become disrupted which allows ethidium homodimer to enter the cells and subsequently bind to the DNA. The stained cell samples may then be viewed and counted under a UV light microscope. This is a good choice of staining necrotic cells as it only stains dead cells, whereas popular ‘cell death staining’ procedures such as TUNEL only stain cells which have undergone programmed cell death (apoptosis).


Calcein-AM/Ethidium Homodimer Assay for Apoptosis/Necrosis transition

This live/dead assay is used to follow the transition between apoptosis and necrosis in real time[4] . Calcein-AM is a fluorescent dye which enters all cells, but only labels live cells. It is enzymatically processed by cytoplasmic enzymes which reside in the cells, resulting in strong green fluorescence. Ethidium homodimer is excluded by live cells with an intact membrane but it enters dead cells and stains their nuclei red. In order to analyse the transition, the cell sample is stained simultaneously by both dyes and the loss of calcein fluorescence and increase in nuclear ethidium homodimer fluorescence are detected with Tert-Butyl Hydroperoxide (tBHP) exposure (Figure 1). The apoptotic cells will initially fluoresce green and as it gradually moves into necrotic cell death, the loss of calcein fluorescence and increase in ethidum homodimer red florescence is observed. Interestingly, there is a period of no fluorescence whatsoever and this presumably corresponds to the lag between loss of calcein fluorescence from the cytosol and breakdown of the plasma membrane, which ultimately allows the entry of ethidium homodimer.



Figure 2. PI and Annexin V double staining

Propidium Iodide (PI)

PI, like its analogue ethidium bromide is a nucleic acid stain as it has a high affinity to DNA and RNA. The substance emits an intense red fluorescence light once it is exposed to UV light and once the dye is bound to the nucleic acids, their fluorescence is enhanced significantly by up to “20-30 fold”[5] . Since PI is membrane impermeant, it is used to identify cells undergoing necrosis in cell culture[6]. When the cell membrane is disrupted, it enters the cell and binds to the DNA and RNA so only necrotic cells fluoresce red. Due to the cell’s extensive membrane damage, necrotic cells are quickly stained with short incubations with PI, whereas apoptotic cells show a much lower PI uptake compared to necrotic cells. You are then able to differentiate between apoptotic (PI dim), healthy (PI –ve) and necrotic (PI bright) cells. These can then be looked at directly under a fluorescence microscope with the fluorescence making them suitable for live cell imaging.


Annexin V/Propidium Iodide assay

A technique used to determine necrotic cells from apoptotic cells is double staining the sample with both Annexin V and Propidium Iodide. Annexin V is a phospholipid binding protein, which binds the membrane lipid phosphatidylserine (PS) that is exposed on the outside of apoptotic cells[7] . However, this technique is not entirely restricted for the detection of apoptotic cells, as annexin V is able to bind the PS on the inside of cell membranes of necrotic cells. Therefore, staining cells with Annexin-V (green fluorescence) in conjunction with propidium iodide (red fluorescence) allows through bivariate analysis the discrimination of apoptotic (Annexin-V positive, PI negative) cells from necrotic (Annexin-V positive, PI positive) cells (Figure 2.)


Current application

The uses of these techniques are extensive; a recent study by Macdonald & Speert (2008) employed the use of necrotic staining to identify necrosis in dendritic cells resulting from a bacterial infection which evolves into cystic fibrosis[8] . Although apoptosis and necrosis are both forms of cells death, they are fundamentally very different processes. To be able to study the morphology, biochemistry and cell-cell interactions of necrotic cells as opposed to apoptotic cells for example, staining techniques such as the Annexin V/Propidium Iodide assay are invaluable as a distinct surface or biochemical marker of necrotic cell death is yet to be identified with the only form of identification being negative markers.



References

  1. Fischer S, Maclean AA, Liu M, Cardella JA, Slutsky AS, Suga M, Moreira JF, Keshavjee S. (2000) Dynamic changes in apoptotic and necrotic cell death correlate with severity of ischemia-reperfusion injury in lung transplantation. Am J Respir Crit Care Med. 162(5):1932-9. PMID: 11069837
  2. Ziegler U, Groscurth P. (2004) Morphological features of cell death. News Physiol Sci.;19:124-8. Review. PMID: 15143207
  3. Xilouri M, Vogiatzi T, Vekrellis K, Park D, Stefanis L. (2009) Abberant alpha-synuclein confers toxicity to neurons in part through inhibition of chaperone-mediated autophagy. PLoS ONE. ;4(5):e5515. PMID: 19436756
  4. Sardão VA, Oliveira PJ, Holy J, Oliveira CR, Wallace KB. (2007) Vital imaging of H9c2 myoblasts exposed to tert-butylhydroperoxide--characterization of morphological features of cell death. BMC Cell Biol. 16;8:11. PMID: 17362523
  5. Unal Cevik I, Dalkara T (2003) Intravenously administered propidium iodide labels necrotic cells in the intact mouse brain after injury. Cell Death Differ.;10(8):928-9. PMID: 12868000
  6. Unal Cevik I, Dalkara T (2003) Intravenously administered propidium iodide labels necrotic cells in the intact mouse brain after injury. Cell Death Differ. ;10(8):928-9. PMID: 12868000
  7. 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.17;184(1):39-51. PMID: 7622868
  8. MacDonald KL, Speert DP. (2008)Differential modulation of innate immune cell functions by the Burkholderia cepacia complex: Burkholderia cenocepacia but not Burkholderia multivorans disrupts maturation and induces necrosis in human dendritic cells. Cell Microbiol.;10(10):2138-49. PMID: 18624797



Lecture 4 - Nucleus

An interesting concept introduced in today’s lecture on the nucleus was the structural support provided by the nuclear cytoskeleton. There are 2 layers of cytoskeleton, the outer, cytoplasmic cytoskeleton being less dense and less organised which surrounds the membrane. The inner layer, consists of intermediate filaments which form the nuclear lamina, a supporting structure which gives the nucleus its shape.


Lecture 5 - Exocytosis

I found the general functions of the Golgi Apparatus quite complex. In particular the organisation and how this subsequently affects the transport of proteins from one cisterna to the next.


Lecture 7 - Mitochondria

Some cellular processes that require a lot of energy from the mitochondria are: cellular metabolism, cellular signalling, protein synthesis and DNA replication.


Lecture 8 - Adhesion

L-CAM: Liver Cell Adhesion Molecule

Ng-CAM: Neuron-glia Cell Adhesion Molecule

N-CAM: Neural Cell Adhesion Molecule

I-CAM: Intercellular Cell Adhesion Molecule


Lecture 10

What is the name of the epidermal layer between the basal and granulosa layer and how does it relate to intermediate filaments?

The epidermal layer between the basal and granulosa layer is called the stratum spinosum. The cells of the stratum spinosum actively synthesize intermediate filaments (cytokeratins). These intermediate filaments are anchored to the desmosomes, which joins adjacent cells providing structural support.


Lecture 14 - Extracellular Matrix 2

2 major different technical methods for generating confocal:

  • 1) a laser scanning confocal microscope (LCSM)
  • 2) a two-photon confocal microscope.

--Mark Hill 23:45, 29 April 2009 (EST) A good try, but not the answer I am looking for,both use a line scanning laser, try again. look at the lab page Confocal Microscopy.

  • laser scanning
  • spinning disk

Lecture 15 - Cell Cycle

What does "S" stand for in the S phase?

The S stage stands for "Synthesis".