Talk:3220823

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  • You have now created your individual project page.


  • Hi there. It was easy and interesting to read. It is well structured and sufficient information on staining necrotic cells. However, there was only one in-text citation in the last section. Maybe consider referencing in any form, Wiki format or APA etc if you can't use Wiki format. Also, is there a link back to the group page???

B) I like the level of detail and research. your referencing is notably absent and you only have a select number of sources. addtionally, there are numerous typos throughout the text... some proofreading might be nescessary. overall it's one of the better one's i've seen. --Peter Kehoe 22:34, 21 May 2009 (EST)

z3189925: Easy to read. Good use of headings. Would have used wiki style referencing and a heading for that sections. Use more relevant and actual references. A section on current research would be good.

Carmen Lam 16:01, 21 May 2009 (EST) Might want to elaborate the purpose of identifying necrotic cells? but overall a good page, good use of images in demonstrating different stains. I agree with the above comment. Maybe you could outline current research which uses this technique, therefore identifying the importance of the technique.

Fantastic, looks like you really know your stuff. Maybe a conclusion with your thoughts about any future directions. Otherwise, well done!

  • I think the page needs more elaboration on the techniques purposes and how it's used in current research. Overall however it was a very informative read.

--Mark Hill 09:57, 20 April 2009 (EST) You have not yet selected your individual project protein/method topic and still missing some homework items. 2009_Student#Individual_Projects Individual Projects

--Mark Hill 09:16, 19 March 2009 (EST) Glad you liked the cytoskeleton, it is a research interest of mine. We will be coming back and discussing the different components of the cell cytoskeleton later in the course. It is has a role during neural cell migration in brain development, where a special basket of microtubules appears to drag the nucleus along with the migrating neural cell body.

--Mark Hill 16:43, 25 March 2009 (EST) Thank you for your feedback. Yes details about Golgi Apparatus transport is quite complex, and not well understood. That is the reason why I showed alternative models for transport from one cisterna to the next.

--Mark Hill 09:41, 3 April 2009 (EST)Correct definitions for CAM terms, lots of science today uses acronyms (because the terms or gene names are very long), but what this can mean is that several similar acronyms can mean different things to different people.

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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. 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 prevents cationic dyes such as PI and trypan blue from entering, and are therefore used for membrane permeability detection. Two of the main live/dead cell assays used in immunochemistry are; ethidium homodimer and PI as they employ this feature of necrotic cells.


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. 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 assayis used to follow through real-time, the transition between apoptosis and necrosis. 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 celcein fluorescence and increase in nuclear ethidium homodimer fluorescence are detected with Tert-Butyl Hydroperoxide (tBHP) exposure. 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 from the cells 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.


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’s 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”. Since PI is membrane impermeant, it is used to identify cells undergoing necrosis in cell culture. 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 [1]. 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.


Refs

[1] A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. J Immunol Methods. 1995 Jul 17;184(1):39-51. PMID: 7622868


Morphological Features of Cell Death U. Ziegler and P. Groscurth Laing, J.; Gober, M.; Golembewski, E.; Thompson, S.; Gyure, K.; Yarowsky P. and Aurelian. L.; "Molecular Therapy" (2006) 13, 870–881.

Dynamic Changes in Apoptotic and Necrotic Cell Death Correlate with Severity of Ischemia–Reperfusion Injury in Lung Transplantation STEFAN FISCHER, ALEXANDRA A. MACLEAN, MINGYAO LIU, JONATHAN A. CARDELLA, ARTHUR S. SLUTSKY, MICHIHARU SUGA, JORGE F. M. MOREIRA, and SHAF KESHAVJEE -(picture link?)