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) []

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) []

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/Application_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**
 * more sensitive than CRA
 * provides a lot more information than CRA
 * can measure number of dead cells just like CRA
 * measurements can be made at the single cell level
 * assay can discriminate between levels of activity that change overtime in the same individual
 * assay can distinguish between NK, T cell, or NKT cell cytotoxicity
 * flow cytometry can detect conjugates of tumor and effectors
 * FCC assay very flexible: cellular activation, cytokine expression, perforin content, signaling molecule expression, absolute numbers, percent specific lysis
 * more flexibility with timing of assay since samples are "fixed" for flow cytometry
 * flow-based assay was measuring kinetics of effector-target interactions with greater efficiency
 * measures a uniformly higher response than CRA


 * Disadvantages of Chromium Release Assay**
 * requirement for considerable numbers of effector cells
 * considerable cost associated with its performance
 * lack of information about behavior of single cells
 * use of radioactivity
 * limited to targets which spontaneously label with 51Cr quantities sufficient for reliable detection
 * many targets are not suitable for CRA because of high spontaneous 51Cr release
 * CRA sensitivity depends on the background, i.e, the amount of 51Cr released from viable target cells over the assay period
 * interassay variability is considerable (CV's over 20% are common)
 * difficulties labeling target cells with 51Cr are common
 * CRA has greater spontaneous release

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

-four-color flow-cytometry-based cytotoxicity assay (FCC)

-lymphokine-activated killer (LAK)

--T cells or NKT (Natural Killer T cells)

-Assay measures NK, T cell, or NKT cell cytotoxicity

-cytotoxicity for defense against: intracellular pathogens, tumor cells, and allogeneic tissue grafts

-chromium release assay from 1968

-samples: melanoma (8) and head and neck cancer (7)

-cell targets: K562 human chronic myelogenous leukemia cells, Daudi, human lymphoma cells (for LAK assay)

-LAK cell generation: IL2

-E:T Ratios

--NK: 50, 25,12,6

--LAK: 6, 3,1.5,0.75

-NK Assay

--Target cells labeled with CellTracker Orange

--FITC-anti-CD16

--FITC-anti-CD56

--ECD-anti-CD3

--Fluorospheres for absolute cell counts

--low flow for conjugate formation

-Stained samples analyzed by flow cytometry within 24 h of staining

-Flow cytometry Instrument: Epics XL-MCL (Beckman Coulter)

-Amnis-based imaging of E and T interactions

--targets identified by cell tracker orange

--aspect ratio close to 1 indicates a round object

--smaller aspect ratios indicate an elongated object, such as that formed by a cluster of two or more cells

-discrimination between effectors, targets, and effector cell-target conjugates in the flow cytometry-based cytotoxicity (FCC) assay is dependent on gating strategies

-7-AAD uptake by live cells is limited and easily distinguished from that of dead cells

-3 h incubation time is optimal for FCC assay

-formation of conjugates precedes lysis and is followed by dissociation of an effector cell from its target

-Conjugate formation at a selected E:T ratio can be measured throughout the cytotoxicity assay' providing information about kinetics of effector and target interactions

-CRA disadvantages

-These data convincingly show that the FCC assay can be substituted for the CRA to measure NK cell and LAK activities without concerns about compairson to historical data sets

-Brightfield Aspect ratio: related to length and width of imaged objects