Flow+cytometry+and+apoptosis+detection+notes+and+paper

This powerpoint/paper was given to us by Tiana.



General notes of the day.. after looking at several papers and protocol:
 * Only saw one paper where 3T3 cells are used. The paper was able to detect killing. However, they used a different DDAO-SE cell tracker, which I think made a difference in detection. See attached - [[file:Flow cytometry 2005_journal of immun.pdf]]
 * Saw that most commonly used cells for control were jurkat, but many other types of cells can be used for this assay as well. We saw that mouse melanoma cells work well. We found a B6 mice cell line but have yet to find one that is Balb C. Have to look for it. Also see the paper above for more cell types.

My notes as I read through it:

Part 1 - A Practical Guide for Detecting Apoptosis by flow cytometry


 * Cells used (for target of effectors? Not clear)
 * Thymocytes - cells of thymus that differentiate into different T cells
 * EL4 mouse thymoma cells - tumor originating from epithelial cells in thymus
 * L1210 cells - mouse lymphocytic leukemia cell line
 * These might be effectors because they are T cells, but they are cancerous... so i'm not sure
 * Suggests it's a good idea to use different dyes to get convincing evidence that apoptosis in indeed occuring as well as information about the nature of the apoptotic pathway.
 * 7-AAD dye - enters cells that have undergone apoptosis.

Part 2 - Multiparametric analysis of apoptosis by flow and immage cytometry


 * combination of assays for apoptosis. Examples:
 * assay that utilizes DNA dyes as plasma membrane permeability indicators (i.e. propidium iodide)
 * I remember seeing this on different papers as one of the axis on the dot plots or histograms
 * assay for mitochondrial membrane potential
 * Annexin V - binds to "flipped" phosphotidylserine (PS)
 * PS is usually inside the lipid membrane (bound), but once the cell undergoes apoptosis, it is found on the outer leaflet of the membrane.
 * Annexin V binds to PS (can only do so when it's outside the membrane.. meaning apoptosis occured)... And Annexin V would have a dye attached to it
 * Caspase activation:
 * One of the earliest measurable markers of apoptosis
 * activation of caspase precedes (in most cases):
 * degradation in cell permeability
 * DNA fragmentation
 * cytoskeletal collapse
 * PS "flipping"
 * likely important in triggering these later manifestations
 * Paper mentions fluorogenic assays for caspase activity that havebeen described in literature
 * OncoImmunin PhiPhiLux system
 * The paper says good things about this.
 * FLICA substrates
 * I feel like i've read some of about these before... can't quite remember what I read though.. Perhaps Kurt remembers, or I could do some more research on this too.
 * The paper describes the integration of this PhiPhiLux caspase substrate system with the annexin V assay and cell membrane permeability to a DNA binding dye.
 * Some materials that could be used:
 * PhiPhiLux system pair with PI or 7-AAD. Excited at 488nm.
 * Reagents are largely non-fluorescent in the uncleaved state and is extremely bright on caspase activation. The apoptotic cells prosses one to three order of magnitude higher fluorescence.
 * Annexin V - should use a DNA binding dye as a cell-permeability indicator too because damaged or necrotic cells with a high degree of membrane permeability will also bind annexin V to their intracellular membrane leaflet.
 * PI is an intercalating DNA binding dye availavle from various sources. Excites at 488nm and emits in 570-630nm range.
 * 7-AAD - DNA binding dye that excited at 488nm and emits in the far red, with peak around 670nm. Can be alternative to PI.
 * There are several options for selecting fluorochrome. For the Beck-Coulter FC500 we have, here are our options:
 * PhiPhiLux-G1D2- FITC channel
 * APC-conjugated annexin V - can be excited with either red diode or He-Ne lasers and emits its signal from PhiPhiLux-G1D2 or the DNA binding dies.
 * PI or 7-AAD: both DNA binding dyes. Can be incorporated into a cell death assay with PhiPhiLux-G1D2 and APC-annexin V.
 * Notes on methods:
 * **don't use more than 0.5-2x10^6 cells per sample, because increasing this number will reduce caspase and annexin labeling efficiency**
 * **Adherent cells pose special challenges for apoptotic analysis due to trauma association with cell dissociation - analysis of adherent state by laser scanning cytometry is preferable to suspension flow cytometry under these circumstances**
 * Cells must be analyzed quickly with this method - they are alive. Analysis must be done immediately after assay.
 * Gating is critical here.
 * See page 150 for the considerations necessary in gating for "early" vs. "late" apoptosis.
 * Looked like the for sure positive peaks were between 10^2-10^3 intensity. The negatives were between 10^0-10^1

Part 3 - Flow cytometry of Apoptosis


 * Have to read that still.