Discussion about research related issues with apoptosis.

• Krishnanand Mishra

  How we can modulate or reprogram one pathway of cell death into another?

  We know that apoptosis and necrosis are required mode of cell death, but in some situation we want to interchange so how we can modulate these or other modalities of cell death into desired pathway.

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  • 
    Patompong Khaw-on replied

    Autophagy contributes to resistance of tumor cells to ionizing radiation. http://www.ncbi.nlm.nih.gov/pubmed/21722986 The role of autophagy in sensitizing malignant glioma cells to radiationAutophagy contributes to resistance of tumor cells to ionizing radiation. http://www.ncbi.nlm.nih.gov/pubmed/21722986 The role of autophagy in sensitizing malignant glioma cells to radiation therapy http://abbs.oxfordjournals.org/content/41/5/341.full

    4 days ago
• Sri Trisha

  Caspase 3/7 activity detection in drug treated cells?

  I would like to test caspase 3/7 activity in jurkat after drug treatment. We purchased the caspase -Glo 3/7 kit from promega. It suggests to use white 96 well plates as it is luminescence assay. 1.I would like to test caspase 3/7 activity in jurkat after drug treatment. We purchased the caspase -Glo 3/7 kit from promega. It suggests to use white 96 well plates as it is luminescence assay. 1. Does anyone has done this assay in clear 96 well plates or black plates? Or its compulsory to do assay in white plates? 2. If we mix Caspase Glo substrate and buffer can we store for a long time?

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    Nick Chacos replied

    You can use clear plates but the signal is approx 10% of that you get in a white plate. You can also use a black plate which reduces the background, thus also enhancing the s/n. Regarding your 2ndYou can use clear plates but the signal is approx 10% of that you get in a white plate. You can also use a black plate which reduces the background, thus also enhancing the s/n. Regarding your 2nd question, you need to review the kit instructions. They should tell you if the substrate mix is stable after dissolving and what conditions to use:4°C,-20°C, etc.

    10 days ago
• Sanjay Vijay

  Why is staurosporine induced apoptosis in cancer cells frowned upon?

  What are better ways of inducing apoptosis?

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  • 
    Gavin Mcstay replied

    You might want to try other chemotherapeutic agents like etoposide, doxorubicin, cisplatin, etc.

    25 days ago
• Sujit Pujhari

  Calculating the percentage of apoptotic cells

  I am doing an apoptosis assay using dual staining with PA and Hoechst. Regarding the calculation percent of apoptotic cells I am little bit confused; Is this procedure right? I have 3 types ofI am doing an apoptosis assay using dual staining with PA and Hoechst. Regarding the calculation percent of apoptotic cells I am little bit confused; Is this procedure right? I have 3 types of population 1. Hoechst stained (bright blue) cells : Apoptotic cells 2. Both PI and Hoechst stained cells : Late stage of apoptosis 3. PI stained cells only (Red cells) : Dead cells Calculation of % of apoptotic cells = Hoechst stained (bright blue) cells + Both PI and Hoechst stained cells / total no of cells - only PI stained cells) x 100

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  • 
    Atish Ray replied

    One important basic to remember in this matter : Please dont be biased in brightness of color: Hoechst / DAPI is practically nothing but the nuclear stain. If apoptotic, you can find lobbedOne important basic to remember in this matter : Please dont be biased in brightness of color: Hoechst / DAPI is practically nothing but the nuclear stain. If apoptotic, you can find lobbed (degraded nucleus) instead intact rounded one in case of non apoptotic control. Annexin V conjugated with flurochrome eg (Ann V-FITC/AnnV CY 3) can also be used to confirm which specifically binds with the exposed phophatidyle serin residue of plasma membrane which expose during early apoptosis .

    Apr 21, 2012
• Vivek Rudrapatna

  Is there any published evidence of Anoikis in invading tumor cells based on the histopathology of tumor biopsies from patients?

  All the literature evidence I seem to find leads back to experimental models but all I'm looking for is a immunohistochemical image of a paraffin-embeded section of a patient biopsy showingAll the literature evidence I seem to find leads back to experimental models but all I'm looking for is a immunohistochemical image of a paraffin-embeded section of a patient biopsy showing TUNEL/ISEL staining of detached, isolated invasive cells. I'm looking for a image to use in a presentation. Can anyone help?

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  • 
    Ashok Sharma replied

    Hi Dear, I don't think so any one published such data even our lab we are also working in more on mechanism like RNA/protein signaling molecules both in vitro or patient sample but not performingHi Dear, I don't think so any one published such data even our lab we are also working in more on mechanism like RNA/protein signaling molecules both in vitro or patient sample but not performing TUNEl/ISEL experiment.

    Apr 12, 2012
• Alex Goldberg

  Does anyone have any technical tips that might help to detect cleaved csp-3 from cell lysates?

  I'm having trouble detecting the p17 band of cleaved caspase-3 through western blot of protein extracted from cells treated with pro-apoptotic compounds, though my full length does decrease due toI'm having trouble detecting the p17 band of cleaved caspase-3 through western blot of protein extracted from cells treated with pro-apoptotic compounds, though my full length does decrease due to treatment.

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  • 
    Dario Caccia replied

    Alex, I used the antibody from cell signaling (#9661) that it works very well. Naturally you need to verify if casp-3 (inactive form) is present in your cell lysate. If your cells don't express theAlex, I used the antibody from cell signaling (#9661) that it works very well. Naturally you need to verify if casp-3 (inactive form) is present in your cell lysate. If your cells don't express the protein, try with other apoptosis assays.

    Apr 11, 2012
• Md. Asaduzzaman Khan

  Poster: Mitochondrial function and cancer (by Nature Reviews)... (free in PDF form)

  http://www.nature.com/nrc/posters/mitochondria/index.html?WT.mc_id=FBK_NatureReviews
• Satheesha Avunje

  Relation between caspase 3 and viral transcription

  I am working on antiviral relative immune expression in flounder, a fish. The virus is a rhabdovirus, called VHSV. I found in the individual host, which expressed high caspase 3 containedI am working on antiviral relative immune expression in flounder, a fish. The virus is a rhabdovirus, called VHSV. I found in the individual host, which expressed high caspase 3 contained significantly low viral mRNA even though it contained very high genomic RNA (negative sense viral genome). Does apoptosis control only viral transcription? Why genomic RNA copies did not decline along with its mRNA?

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  • 
    Satheesha Avunje replied

    Thanks I got the pdf

    Mar 14, 2012
• Heba Emam

  How does Hyperthermia affect the cell membrane, especially of cancer cells?

  If Hyperthermia induces cell death, does the cell membrane have any role in that?

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• Aswartha Harinathreddy

  How can I detect caspase-3 in cells?

  Any method to find caspase 3 in cells?

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  • 
    Justin Rothfuss replied

    The Promega Kit - Apo-ONE Homogeneous Caspase-3/7 Assay works well. It can detect caspase-3 activity in cells plated in 96-well format. Promega also amkes another kit similar to the one above thatThe Promega Kit - Apo-ONE Homogeneous Caspase-3/7 Assay works well. It can detect caspase-3 activity in cells plated in 96-well format. Promega also amkes another kit similar to the one above that detects active caspase-3 in cells, just can't remember the name right now. Peace.

    Mar 2, 2012
• Aswartha Harinathreddy

  Is it is possible to identity the capases 3, 9 ,8, by only SDS PAGE analysis?

  Is it is possible to identity the capases 3, 9 ,8, by only SDS PAGE analysis?

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• Tal Etzyoni

  Hi, I am trying to detect Apoptosis by Facs (AnnexinnV) and by western blot (Cleaved caspase 3) and want to cross their appearance. However, I could not find in the literature the time line. I know AnnexinV appears at the early stage of apoptosis. Caspase3 cleavage is also in the early± stages. Isn't it? So, if I want to find both markers, what comes first? TNX Tali

  Apoptosis

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  • 
    Shi Liu replied

    The classic approach is the live staining with AnnexinV plus PI (flow cytometry or epifluorescence microscopy). In the early stage of apoptosis (seconds- a couple of minutes) you will see onlyThe classic approach is the live staining with AnnexinV plus PI (flow cytometry or epifluorescence microscopy). In the early stage of apoptosis (seconds- a couple of minutes) you will see only AnV-positive, followed by AnV-postive and PI-positive during the progress of apoptosis. The time course is probably cell-specific. Western blot analysis is not sensitive to detect the early events.

    Feb 29, 2012
• Patrick Kao

  Tunel assay

  Which TUNEL assay do you recommend for IHC staining?

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  • 
    Koichi Ito replied

    Click-iT TUNEL Imaging kit from Invitrogen works really well with tumour sections. It's a bit expensive but worth trying.

    Feb 24, 2012
• Johana Guevara

  I would like to know which method could be better for evaluating apoptosis in tissue?

  I need to evaluate apoptosis in a cell culture in a scaffold, and I can't use flow cytometry since the cells are very difficult to disgregate. And I have no experience in this... somebody could helpI need to evaluate apoptosis in a cell culture in a scaffold, and I can't use flow cytometry since the cells are very difficult to disgregate. And I have no experience in this... somebody could help me?

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  • 
    Muhammad Yasar replied

    Dear Johana, This review article contains different methods of detecting apoptosis in tissue. Have a look once.... http://www.histochem.org/archives/1101.pdf

    Feb 22, 2012
• William Jackson

  Is cancer a metabolic (mitochondrial) disorder?

  During the first half of the last century Otto Warburg proposed the idea that cancer was linked to a disorder in the mitochondria which prevented apoptosis. Therapists like Max Gerson and JoannaDuring the first half of the last century Otto Warburg proposed the idea that cancer was linked to a disorder in the mitochondria which prevented apoptosis. Therapists like Max Gerson and Joanna Budvig made promising clinical progress using this approach. A review of the idea may be found here: http://www.nature.com/nrclinonc/journal/v3/n7/full/ncponc0538.html If true: then ingesting cytotoxic chemicals, whilst trying to promote autophagy and apoptosis on the other hand, is counterproductive.

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