Flow cytometry and apoptosis detection notes and paper

2015-01-13

azim58 - Flow cytometry and apoptosis detection notes and paper

This powerpoint/paper was given to us by Tiana.

Flow cytometry from Tiana - Part 1.pdf
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-19-2014d1132\01-19-2014d1201\Flow cytometry from Tiana - Part 1.pdf"
Flow cytometry from Tiana - Part 2.pdf
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-19-2014d1132\01-19-2014d1201\Flow cytometry from Tiana - Part 2.pdf"
Flow cytometry from Tiana - Part 3.pdf
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-19-2014d1132\01-19-2014d1201\Flow cytometry from Tiana - Part 3.pdf"

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 - Flow
cytometry 2005_journal of immun.pdf
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-19-2014d1132\01-19-2014d1201\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)

o Thymocytes - cells of thymus that differentiate into different T
cells
o EL4 mouse thymoma cells - tumor originating from epithelial cells
in thymus
o L1210 cells - mouse lymphocytic leukemia cell line
o 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:

o 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

o assay for mitochondrial membrane potential
o 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:

o One of the earliest measurable markers of apoptosis
o activation of caspase precedes (in most cases):
- degradation in cell permeability
- DNA fragmentation
- cytoskeletal collapse
- PS "flipping"

o likely important in triggering these later manifestations

Paper mentions fluorogenic assays for caspase activity that havebeen

described in literature
o OncoImmunin PhiPhiLux system
- The paper says good things about this.

o FLICA substrates
o 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:

o 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.

o 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.
o PI is an intercalating DNA binding dye availavle from various
sources. Excites at 488nm and emits in 570-630nm range.
o 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:
o PhiPhiLux-G1D2- FITC channel
o 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.
o 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:

o don't use more than 0.5-2x10^6 cells per sample, because
increasing this number will reduce caspase and annexin labeling
efficiency
o 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
o Cells must be analyzed quickly with this method - they are alive.
Analysis must be done immediately after assay.

Gating is critical here.

o See page 150 for the considerations necessary in gating for
"early" vs. "late" apoptosis.
o 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.