The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity

M. A. Gillissen; E. Yasuda; G. de Jong; S. E. Levie; D. Go; H. Spits; P. M. van Helden; M. D. Hazenberg

doi:https://doi.org/10.1016/j.jim.2016.04.002

The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity

M. A. Gillissen, E. Yasuda, G. de Jong, S. E. Levie, D. Go, H. Spits, P. M. van Helden, M. D. Hazenberg

Research output: Contribution to journal › Article › Academic › peer-review

29 Citations (Scopus)

Abstract

Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay

Original language	English
Pages (from-to)	16-23
Journal	Journal of immunological methods
Volume	434
DOIs	https://doi.org/10.1016/j.jim.2016.04.002
Publication status	Published - 2016

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https://doi.org/10.1016/j.jim.2016.04.002

Cite this

@article{dccc0e4e63a84f03ae1bce3e135ba19d,

title = "The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity",

abstract = "Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay",

author = "Gillissen, {M. A.} and E. Yasuda and {de Jong}, G. and Levie, {S. E.} and D. Go and H. Spits and {van Helden}, {P. M.} and Hazenberg, {M. D.}",

year = "2016",

doi = "https://doi.org/10.1016/j.jim.2016.04.002",

language = "English",

volume = "434",

pages = "16--23",

journal = "Journal of immunological methods",

issn = "0022-1759",

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T1 - The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity

AU - Gillissen, M. A.

AU - Yasuda, E.

AU - de Jong, G.

AU - Levie, S. E.

AU - Go, D.

AU - Spits, H.

AU - van Helden, P. M.

AU - Hazenberg, M. D.

PY - 2016

Y1 - 2016

N2 - Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay

AB - Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay

U2 - https://doi.org/10.1016/j.jim.2016.04.002

DO - https://doi.org/10.1016/j.jim.2016.04.002

M3 - Article

C2 - 27084117

SN - 0022-1759

VL - 434

SP - 16

EP - 23

JO - Journal of immunological methods

JF - Journal of immunological methods

ER -