High‐Throughput Cytotoxicity Screening by Propidium Iodide Staining

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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

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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

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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

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Figures

Figure 9.24.1 Flow cytometry analysis parameters for cytotoxicity assay. Plots of forward scatter (Lin FS) versus red propidium iodide (PI) cell fluorescence (Log PI) allow distinction of viable cells (rectangular gate labeled “live”) and dead cells with damaged membranes that take up PI (rectangular gate labeled “PI⁺ ”). Illustrated are composite linear forward scatter versus log PI fluorescence intensity dot‐plot data from (A) an entire 384‐well plate, (B) a single negative control well, and (C) a single positive control well. The cell numbers detected in each gate are indicated for the two control wells in panels B and C . Also illustrated are PI fluorescence intensity histograms for (D) cells in the live‐cell gate of the negative control well and (E) cells in the PI⁺ cell gate of the positive control well.

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Figure 9.24.2 Time‐resolved analysis of individual wells. Cells were cultured 24 hr at 37°C in the presence of test compounds at a final concentration of 1 µM. Illustrated are cytotoxicity assay results from rows L through P (120 wells) of a 384‐well plate. Cell numbers detected in each well appear as 24 separate peaks resolved over a time span of ∼40 sec per row. Cell numbers detected in the dead‐cell gate (PI⁺ , top panel) and the live‐cell gate (Lv, bottom panel) are shown for each row. Rectangles represent left and right boundaries on the time axis encompassing data from each well (upper and lower rectangle boundaries are only to serve as visual aids). All wells in columns 2 and 23 contained DXR as a positive control that killed >95% of cells. Three test compounds that killed >50% of cells (indicated by arrows at wells M20, N9, and P22) were considered cytotoxic hits. Five additional compounds (wells M16, O6, P5, P6, and P14) were also determined to be cytotoxic after 72 hr incubation in a separate plate prepared and analyzed in parallel (data not shown).

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Literature Cited

Literature Cited
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High‐Throughput Cytotoxicity Screening by Propidium Iodide Staining

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited