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    Apoptosis Induction

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    hemlata09
    The document describes two general methods for inducing apoptosis in cells: biological induction using anti-Fas monoclonal antibodies or TNF ligands, and chemical induction using DNA damaging agents like doxorubicin, 5-fluorouracil, paclitaxel, or vinblastine. For biological induction, anti-Fas mAb is added to Jurkat cells and incubated for 3-6 hours to induce apoptosis, which is then detected. For chemical induction, cells are treated with DNA damaging agents for 40-48 hours to induce p53-dependent apoptosis, assessed by morphology and viability changes.

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    Apoptosis Induction

    Uploaded by

    hemlata09
    29 views2 pages
    The document describes two general methods for inducing apoptosis in cells: biological induction using anti-Fas monoclonal antibodies or TNF ligands, and chemical induction using DNA damaging agents like doxorubicin, 5-fluorouracil, paclitaxel, or vinblastine. For biological induction, anti-Fas mAb is added to Jurkat cells and incubated for 3-6 hours to induce apoptosis, which is then detected. For chemical induction, cells are treated with DNA damaging agents for 40-48 hours to induce p53-dependent apoptosis, assessed by morphology and viability changes.

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    The document describes two general methods for inducing apoptosis in cells: biological induction using anti-Fas monoclonal antibodies or TNF ligands, and chemical induction using DNA damaging agents like doxorubicin, 5-fluorouracil, paclitaxel, or vinblastine. For biological induction, anti-Fas mAb is added to Jurkat cells and incubated for 3-6 hours to induce apoptosis, which is then detected. For chemical induction, cells are treated with DNA damaging agents for 40-48 hours to induce p53-dependent apoptosis, assessed by morphology and viability changes.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    29 views2 pages

    Apoptosis Induction

    Uploaded by

    hemlata09
    The document describes two general methods for inducing apoptosis in cells: biological induction using anti-Fas monoclonal antibodies or TNF ligands, and chemical induction using DNA damaging agents like doxorubicin, 5-fluorouracil, paclitaxel, or vinblastine. For biological induction, anti-Fas mAb is added to Jurkat cells and incubated for 3-6 hours to induce apoptosis, which is then detected. For chemical induction, cells are treated with DNA damaging agents for 40-48 hours to induce p53-dependent apoptosis, assessed by morphology and viability changes.

    Copyright:

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    Download as PDF, TXT or read online from Scribd
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    of 2

    General protocols for inducing apoptosis in cells

    Apoptosis may be induced in experimental systems through a variety of methods. In general, they can be divided
    into 2 categories: a) biological induction; and b) chemical induction.

    A) Biological induction of apoptosis

    Activation of either Fas or TNF-receptors by the respective ligands or by cross-linking with agonist antibody
    induces apoptosis of Fas- or TNF receptor-bearing cells. Below is general protocol used to induce apoptosis using
    anti-Fas mAb in Jurkat Cells.

    1. Grow Jurkat cells in RPMI-1640 medium containing 10% fetal bovine serum in a humidified, 5% CO2
    incubator at 37°C.
    5
    2. Suspend the cells in fresh medium at a concentration of 1 × 10 cells/ml. After two to three days of
    incubation in a 37°C, 5% CO2 incubator, harvest the cells by centrifugation at 300–350 × g for 5 mins.
    5
    3. Resuspend cells in fresh medium to 5 × 10 cells/ml and add anti-Fas mAb to a final concentration of 0.05–
    0.1 µg/ml. Incubate for 3–6 hours in a 37°C incubator. As a negative control, incubate untreated cells (no
    anti-Fas mAb) under the same conditions. (Stop here for homogeneous assay, or plate the cells in a 96-
    well plate.)
    4. Harvest the cells by centrifugation at 300–350 × g for 5 mins.
    5. Remove all medium and resuspend cells in PBS.
    6
    6. Repeat centrifugation and resuspend the cell pellet in PBS to 1.5 × 10 cells/ml.
    7. Proceed to apoptosis detection.

    B) Chemical induction of apoptosis

    Depending on the agent selected and the concentrations used, maximal induction of a particular protein may occur
    within 8 to 72 hours post-treatment. However, not all proteins are affected by reagents in a particular cell line. The
    following protocol is based on p53-dependent G1-arrest that occurs in response to DNA damage by chemical
    agents. A typical time course for p53 induction is 40 to 48 hours treatment with a DNA damaging agent.

    2
    1) Inoculate each of 2 or more 10 cm tissue culture dishes for adherent cells or T-75 flasks for non-adherent cells
    6
    with approximately 1 x 10 cells. One dish or flask will be used as negative control for non-induced or basal level
    expression.

    2) Confirm that cells are growing by visual inspection of tissue culture dishes or by viable cell counts on non-
    adherent cells in T-75 flasks. Add DNA damaging agents to recommended final concentrations. The list below
    gives suggestions of final concentrations that can be used for several well known apoptosis indicuing chemicals:

    Examples:

    0.2 µg/ml Doxorubicin (stock prepared in H2O, 25 µg/ml)

    5-Fluorouracil (stock prepared in DMSO)

    100-58 nM Paclitaxel (stock prepared in DMSO)

    www.abcam.com/technical
    60 nM Vinblastine (stock prepared in methanol)

    1 mM staurosporine in DMSO

    Add appropriate volume of buffer or solvent (i.e. DMSO) to the non-induced control.

    3) Check cells to determine if cells have begun to apoptose. This can be assessed by checking the morphology of
    the cells (cells will become granulated and blebbing may be observed). Viability can be checked using eg trypan
    blue cell counting. Harvest cells if greater than 75% of the cells appear to have died upon trypan blue viability
    counting.

    4) Harvest cells and prepare lysates for either Western blotting or immunoprecipitation. For any agent used, a time
    course of induction can be performed by inoculating additional dishes or flasks and harvesting at various times (i.e.
    24, 48 and 72 hours) after addition of the DNA damaging agent. For the examination of apoptotic proteins, dead
    cells should also be collected. Always compare levels of p53 from treated cells and controls to confirm induction.

    www.abcam.com/technical

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