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    Analyzing apoptotic cell/tissue by Flow Cytometry

    The following protocol is suitable for visualizing cells or tissue harvested from an in vivo study  and analyzed by flow cytometry. These protocols are for guideline only and we recommend following the instructions for use provided with each product as concentration, preparation and excitation/emmission criteria may vary between each product. 

    Principle of the CAS-MAP™ assays and probes

    The CAS-MAP™ apoptosis assays and probes are cell permeable reagents for detecting active caspases in cell culture, tissues and animal studies. CAS-MAP™ is either added to cell culture or is injected intravenously (IV) and allowed to circulate. The low molecular weight and characteristics of the probe allow it to penetrate the cell membrane and the blood brain barrier/blood retinal barrier. Once the probe is inside the cell, it forms a covalent bond with the active catalytic site of caspases, which tags the active caspase with a fluorescent label. The bound reagent remains inside the cell membrane of apoptotic cells, while unbound reagent is removed from non-apoptotic cells by a wash step in cell culture or the circulatory system, kidneys and liver in vivo. Apoptotic cells have more active caspase than control cells and therefore fluoresce brighter when labeled with the caspase probe.

    Cell Culture Experimental Set-up:

    Culture cells as required to induce apoptosis. The density of the cultured cells should not exceed 106 cells/ml. Typically, 106 cells are aliquoted into wells of a 24 well culture plate in a final volume of 1 ml. Investigators must optimize the conditions and kinetics for induction of apoptosis in their system. A control group of cells not induced to undergo apoptosis should also be cultured. It is recommended that induced and non-induced cultures be set up for each labeling condition.

    Note: If using adherent cells, remove cells using slight scraping, tituration or trypsinization as appropriate for your cell line. Since apoptotic cells may detach, it is recommended that the culture media be harvested and combined with the adherent cells. If using trypsinization, neutralize with a trypsin inhibitor. Cell pellets should be resuspended to 1 mL and counted prior to staining.

    During the last 15-30 minutes of apoptosis induction, add 10 µL of CAS-MAP™ working solution per 1 mL culture volume. The staining is performed at 37° C. After the staining period, harvest the cells into 5 mL tubes, centrifuge at 500 x g for 2 minutes and wash twice with 3 mL of 1X Wash Buffer

    In Vivo Experimental Set-up:

    Plan your in vivo experiment so that you can inject CAS-MAP™ at the time when you expect apoptosis to be occuring. A pilot experiment may be required to determine the timing and dose of CAS-MAP™ to inject as the resulting positive fluorescent signal is a direct measure of caspase activity at the time of the injection.

    1. Expose the animals to the experimental condition to induce apoptosis or caspase activity in the target tissue. Be sure to include positive and negative control groups.
    2. At the desired time, prepare the amimals for intravenous injection.
    3. Inject 100 uL of 1x CAS-MAP™ into the tail vein (or other IV administration site). If you need a larger injection volume, dilute theCAS-MAP with additional injection buffer and inject 1/6 of the total volume into each animal. 
    4. AllowCAS-MAP to circulate within the animal for at least 30 minutes. At 60 minutes, most of the unboundCAS-MAP will be cleared from the blood stream. Generally, the longer the reagent circulates, the lower the background signal. However, apoptotic cells will be lost over time.CAS-MAP will remain inside an apoptotic cell as long as the cell membrane is intact.
    5. Terminate the animal at the appropriate time and prepare tissues for flow cytometry.

    Preparing tissues:

    Extract the tissue, create a single cell suspension and perform any additional staining required. Use a method that will keep cell membranes intact as much as possible. Samples may be fixed, however it is preferred to analyze immediately after staining.

    Flow cytometry:

    Observe the cells on a SS versus FS linear dot plot and identify and gate on cells of interest. Apoptotic cells have been described to undergo changes in their light scattering properties. Make sure that the scatter gate used to identify cells of interest will accommodate these changes. Flourescence detection is observed by employing the appropriate laser excitation for the CAS-MAP™ probe being used. Generate histograms depicting FL1 (x-axis) versus cell count (y-axis). Make regions of the histogram that identify positive and negative events. The caspase positive cell population will appear as a separate peak or as a shoulder of the first peak demonstrating increased fluorescence intensity.

    Figure 1: Imaging with CAS-MAP™ Green probe to assess apoptosis in bone marrow. Animals were injected with CAS-MAP™ 45 minutes prior to termination. Bone marrow cells were obtained and analyzed by flow cytometry.