High‐Throughput Cytotoxicity Screening by Propidium Iodide Staining

Bruce S. Edwards1, Irena Ivnitski‐Steele1, Susan M. Young1, Virginia M. Salas1, Larry A. Sklar1

1 University of New Mexico, Albuquerque, New Mexico
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.24
DOI:  10.1002/0471142956.cy0924s41
Online Posting Date:  July, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes a system for the automated high‐throughput analysis of cell cytotoxicity in 96‐well and 384‐well microplates. Discrete cell cultures are analyzed at rates of 40/min (∼2.5 min/96 wells, ∼10 min/384 wells) and cytotoxicity is quantified on the basis of a combination of propidium iodide (PI) fluorescence analysis and cell counting performed by the flow cytometer. Only 2 µl is aspirated from a culture for analysis so that assays can be performed in small volumes to minimize reagent cost and usage.

Keywords: high‐throughput screening; cytotoxicity; propidium iodide; cell viability assay

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Cytotoxicity Screening in 384‐Well Format with Automated Liquid Handling
  • Alternate Protocol 1: Cytotoxicity Screening in 96‐Well Format with Hand Pipetting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Cytotoxicity Screening in 384‐Well Format with Automated Liquid Handling

  Materials
  • CCRF‐CEM cells (ATCC #CCL‐119), >95% viability determined by eosin exclusion (Hoskins et al., ) on a representative sample: passaged twice weekly in T175 tissue culture flasks at a cell density of 0.5 to 2 × 106 viable cells/ml (see appendix 3B; quality of assay not affected by passage number)
  • Culture medium (see recipe): 950 µl/plate
  • Culture medium with 5% DMSO (see recipe): 750 µl/plate
  • 1 mM doxorubicin (DXR; Sigma) in dimethyl sulfoxide (DMSO; Sigma): 50 µl/plate
  • DMSO (Sigma)
  • 0.05 mg/ml propidium iodide (PI) in phosphate‐buffered saline (PBS; appendix 2A): 800 µl per plate
  • Compounds for cytotoxicity testing (1 to 10 mM in DMSO) in a stock compound microplate: prepared by dispensing test compounds into columns 3 through 22 of a 384‐well polypropylene plate (using a Biomek NX/384 multichannel pipettor) and covering with aluminum foil adhesive covers until use
  • 384‐well polypropylene, shallow profile, conical‐bottom microplates (Greiner): sterilized for at least 10 hr under a 253.7‐nm germicidal lamp in a biological safety cabinet; 3 plates/320 wells of test compounds
  • Liquid‐handling equipment including:
    • MapC2 dispenser (Titertek)
    • Biomek NX/384 multichannel pipettor (Beckman Coulter)
    • Biomek NX Span8 (Beckman Coulter)
    • 50‐ml conical centrifuge tubes, sterile
  • Biomek Seal and Sample aluminum foil adhesive well plate covers (Beckman Coulter): two seals/384‐well stock compound plate
  • Sterile lids for plates (e.g., Costar)
  • HyperCyt with Dako‐Cytomation Cyan flow cytometer (Dako)
  • Additional reagents and equipment for counting (appendix 3a) and culturing ( appendix 3B) cells

Alternate Protocol 1: Cytotoxicity Screening in 96‐Well Format with Hand Pipetting

  • 96‐well, conical‐bottom PCR plate (ISC BioExpress): sterilized for at least 10 hr under a 253.7‐nm germicidal lamp in a biological safety cabinet
  • 12‐channel multichannel pipettors, 0.5 to 10 µl and 10 to 100 µl
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bugelski, P.J., Atif, U., Molton, S., Toeg, I., Lord, P.G., and Mogan, D.G. 2000. A strategy for primary high‐throughput cytotoxicity screening in pharmaceutical toxicology. Pharm. Res. 17:1265‐1272.
   Edwards, B.S., Kuckuck, F., and Sklar, L.A. 1999. Plug flow cytometry: An automated coupling device for rapid sequential flow cytometric sample analysis. Cytometry 37:156‐159.
   Edwards, B.S., Young, S.M., Oprea, T.I., Bologa, C., Prossnitz, E., and Sklar, L.A. 2006. Biomolecular screening of formylpeptide receptor ligands with a sensitive, quantitative, high‐throughput flow cytometry platform. Nat. Protoc. 1:59‐66.
   Hoffman, A.F. and Garippa, R.J. 2007. A pharmaceutical company user's perspective on the potential of high content screening in drug discovery. Methods Mol. Biol. 356:19‐31.
   Hoskins, J.M., Meynell, G.G., and Sanders, F.K. 1956. A comparison of methods for estimating the viable count of a suspension of tumor cells. Exp. Cell. Res. 11:297‐305.
   Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F., and Riccardi, C. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139:271‐279.
   Nolan, J.P., Lauer, S., Prossnitz, E.R., and Sklar, L.A. 1999. Flow cytometry: A versatile tool for all phases of drug discovery. Drug Discov. Today 4:173‐180.
   O'Brien, P. and Haskins, J.R. 2007. In vitro cytotoxicity assessment. Methods Mol. Biol. 356:415‐425.
   Ormerod, M.G., Sun, X.M., Brown, D., Snowden, R.T., and Cohen, G.M. 1993. Quantification of apoptosis and necrosis by flow cytometry. Acta Oncol. 32:417‐424.
   Ramirez, S., Aiken, C.T., Andrzejewski, B., Sklar, L.A., and Edwards, B.S. 2003. High‐throughput flow cytometry: Validation in microvolume bioassays. Cytometry A 53:55‐65.
   Schmidt, I., Krall, W.J., Uittenbogaart, C.H., Braun, J., and Giorgi, J.V. 1992. Dead cell discrimination with 7‐amino actinomycin‐D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry 13:204‐208.
   Tuschl, H. and Schwab, C.E. 2004. Flow cytometric methods used as screening tests for basal toxicity of chemicals. Toxicol. in Vitro 18:483‐491.
   Tuschl, H. and Schwab, C.E. 2005. The use of flow cytometric methods in acute and long‐term in vitro testing. Toxicol. in Vitro 19:845‐852.
   Walum, E., Hedander, J., and Garberg, P. 2005. On the relevance of cytotoxicity measurements, barrier passage determinations and high‐throughput screening in vitro to select potentially hazardous compounds in large sets of chemicals. Toxicol. Appl. Pharmacol. 207:393‐397.
   Yeh, C.G., His, B., and Faulk, W.P. 1981. Propidium iodide as a nuclear marker in immunofluorescence. II: Use with cellular identification and viability studies. J. Immunol. Methods 43:269‐275.
   Zhang, J.H., Chung, T.D., and Oldenburg, K.R. 1999. A simple statistical parameter for use in evaluation and validation of high‐throughput screening assays. J. Biomol. Screen. 4:67‐73.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library