Multiparametric Flow Cytometry 2007

2015-01-13

azim58 - Multiparametric Flow Cytometry 2007


Title:
A novel multiparametric flow cytometry-based cytotoxicity assay
simultaneously immunophenotypes effector cells: Comparisons to a 4 h
51Cr-release assay (2007)
http://www.sciencedirect.com/science/article/pii/S0022175907001779

This article was cited by 22 other articles (as revealed by Scopus)

Original Paper:

"C:\kurt\storage\CIM Research Folder\DR\2013\1-28-13\1002\Multiparametric
Flow Cytometry CTL 2007.pdf"



Annotated Paper:

"C:\kurt\storage\CIM Research Folder\DR\2013\1-28-13\1002\A novel
(annotated) multiparametric flow cytometry-based cytotoxicity assay
simultaneously immunophenotypes effector cells Comparisons to a 4 h
Cr-release Assay.pdf"




Comment on Paper:
This FCC assay is looking really good to me.

When I first started reading about flow cytometry cytotoxicity assays
such as the caspase assay, I thought the caspase assay is a useful assay
but perhaps still not as powerful as the chromium release assay. Although
T cells almost "invariably" use the caspase pathway to kill cells, there
are still other ways that T cells can kill a cell which are not fully
understood at this time (Measurement of CTL-Induced Cytotoxicity The
Caspase 3 Assay (2003)). A T cell may be forced to use such alternative
cytotoxicity pathways if a cell has a mutation in a gene such as Apaf-1.
Although these issues look unlikely to negatively affect any of our
assays, there might be some chance.

However, after reading about the caspase flow cytometry assay, I came
across this 2007 flow cytometry paper.

A novel multiparametric flow cytometry-based cytotoxicity assay
simultaneously immunophenotypes effector cells: Comparisons to a 4 h
51Cr-release assay (2007) (cited 22 times since)
http://www.sciencedirect.com/science/article/pii/S0022175907001779

This assay looks very good since it is more sensitive than the chromium
release assay, provides so much more information, is a very flexible
test, and it is not just focused on one pathway. In fact, this assay can
measure the conjugation of an effector cell (NK, T, or NKT cell) with a
target cell. Therefore, regardless of the pathway used, one can detect
whether effector cells are conjugating with target cells and if they are
killing them.

These flow cytometry assays seem to be gaining in popularity, and provide
a lot more information than the chromium assay. Another paper that I
haven't had too much time to look at yet uses such methods to test cancer
vaccines.

Application of a Flow Cytometric Cytotoxicity Assay for Monitoring Cancer
Vaccine Trials (2009)
http://journals.lww.com/immunotherapy-journal/Abstract/2009/02000/Applicati
on_of_a_Flow_Cytometric_Cytotoxicity.11.aspx

Are we completely sure that we want to buy a new scintillation counter? I
think people in our lab would be less hesitant to pursue these
cytotoxicity experiments if they don't have to work with radiation. Flow
cytometry is also a very common method so people would be able to teach
us and let us use their equipment. Since the flow cytometry methods are
so sensitive, powerful, and informative, perhaps we can just use those
instead of the chromium release assay. Or perhaps we could start with
flow cytometry assays and then move to chromium release assays if
necessary later on.

Notes:
Advantages of Four Color Cytometry

o can measure number of dead cells just like CRA
o measurements can be made at the single cell level
o assay can discriminate between levels of activity that change
overtime in the same individual
o assay can distinguish between NK, T cell, or NKT cell cytotoxicity
o flow cytometry can detect conjugates of tumor and effectors
o FCC assay very flexible: cellular activation, cytokine
expression, perforin content, signaling molecule expression, absolute
numbers, percent specific lysis

flow cytometry
interactions with greater efficiency

Disadvantages of Chromium Release Assay

sufficient for reliable detection
51Cr release
released from viable target cells over the assay period

Phenotypic Characteristics of Cells
NK: CD3-CD16+CD56+
T: CD3+CD16-CD56-
NKT: CD3+CD16+CD56+





and allogeneic tissue grafts



human lymphoma cells (for LAK assay)

















by a cluster of two or more cells

conjugates in the flow cytometry-based cytotoxicity (FCC) assay is
dependent on gating strategies

of dead cells



of an effector cell from its target

the cytotoxicity assay' providing information about kinetics of effector
and target interactions


the CRA to measure NK cell and LAK activities without concerns about
compairson to historical data sets