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measurement of green fluorescent proteins and a red fluorescent protein in live cells in the Analyst GT multimode reader

Evelyn McGown, Ph.D., Jinfang Liao, M.D., Ph.D., Mary Kassinos, Irina Osetinsky and Jayne Hesley, Molecular Devices Corporation, 1311 Orleans Dr., Sunnyvale, CA 94089.

Introduction
Fluorescent proteins have become enormously popular as tools for monitoring biological events in vivo. They can be cloned in a diverse range of cells and organisms, from bacteria and yeast, to plants and mammals. The fluorescent proteins are stable, have minimal toxicity and have the ability to generate visible fluorescence in vivo without the need for external cofactors. Thus they can be used as molecular tags or as independent reporters to visualize, track and quantify many different cellular processes, including protein synthesis and turnover, protein translocation, gene induction and cell lineage.

The various proteins have different colors, so they can be used in multiplexed assays. They can be monitored by fluorescent microscopy and flow cytometry. If there is no need to physically sort the cells or to monitor intracellular migration, microplate fluorometry offers a preferable, more convenient and higher-throughput detection system. Below, we show that the Analyst GT multimode reader from Molecular Devices can easily and noninvasively measure fluorescent proteins in living cells.

We obtained three HEK-293 cell lines from BD Biosciences Clontech, each stably transfected with a different fluorescent protein. The purposes of the study were: 1) to determine the optimal instrument settings for three cell lines, and 2) to prepare dilution series of each cell line to estimate lower limits of detection (LLD).


MATERIALS
HEK-293 Cell Lines stably expressing fluorescent proteins were obtained from BD Biosciences Clontech:
AcGFPa variant of GFP cloned from Aequorea coerelescens (a jellyfish distinct from victoria)
ZsGreensimilar to GFP, but b righter (from reef coral)
DsReda red-shifted fluorescent protein cloned from reef coral

HEK-293 Cell line non-transfected (ATCC Cat. #CRL-1573)

DME: High Glucose (Irvine Scientific Cat. #9024)

G418 (Geneticin, Gibco/Invitrogen Cat. #11811-031)

FBS (Irvine Scientific Cat. #3000A)

Glutamine/Pen/Strep Solution (Gibco/ Invitrogen Cat. #10378-016)

Trypsin/EDTA 1X in HBSS (Irvine Scientific Cat. #9341)

HBSS Buffer (1X Hanks Balanced Salt Solution with 20 mM HEPES buffer): Made from 10X Hanks Balanced Salt Solution (100 mL), 1M HEPES (20 mL) and cell culture treated water (880 mL)

10X Hanks Balanced Salt Solution (Gibco/ Invitrogen Cat. #14065-056)

1M HEPES (Irvine Scientific Cat. #9319)

Water for cell culture (Irvine Scientific Cat. #9312)

Black-Wall Clear-Bottom 96-Well Microplate (Costar Cat. #3603)

Black-Wall Clear-Bottom 384-Well Microplate (Costar Cat. #3712)

Analyst GT Multimode Reader (Molecular Devices)


METHODS
Cell preparation and analysis
The cells were cultured in bulk flasks in DME + 10% FBS + 1% Pen/Strep/L-glutamine + 500 g/mL of G418. Non-transfected HEK cells were included as controls. The night before the experiment, they were trypsinized and the cell suspension was diluted serially to give cell densities ranging from 500,000 down to 100 cells per mL. They were seeded overnight in 96-well (100 L/well) and 384-well microplates (25 L/well). Thus the seeded cell densities were 50,000 to 10 cells/well (96-well plate) and 12,500 to 2.5 cells/well (384-well plate). There were 12 replicate wells per dilution in both 96well and 384-well plates. The microplates were read the next day from the bottom and the top in the Analyst GT multimode reader.

Analyst GT settings
Published values1 for the Excitation and Emission lambda maxima of the three proteins are given in Table 1.

In a preliminary experiment, we determined the optimal filter configurations to be:
AcGFP and ZsGreen: 485/510 (485- 20 nm/530-25 nm gave inferior results)

DsRed: 550/590 (550-10 nm/580-10 nm gave inferior results)

The Analyst GT parameters in the present study are summarized in Table 2.


RESULTS
The results from the dilution series in a 384-well plate are shown in Figure 1 (bottom-read) and Figure 2 (top-read). Similar plots were obtained for the 96-well plates (not shown). The ZsGreen cell line (upper curve) was approximately 3.5 times brighter than the other two. Although the DsRed cell line was dimmer than the Zs-Green, their limits of detection were similar because the background was lower at the DsRed wavelengths. (See Table 3.)



* Values are expressed in terms of #cells/well. To convert to #cells/mm2, divide the 96-well values by 129 mm2 /well and the 384 values by 25 mm2/well.

Two 96-well experiments were performed and the results of the first experiment are included in parentheses.


In general, the plots were slightly nonlinear, dropping off at the upper end. We speculate that this might be due to the tendency of cells at high density to migrate up the microplate walls and therefore be out of the beam.

Table 3 gives the estimated lower limits of detection (LLD) for each of the cell lines in two separate experiments. (The first experiment was done with 96-well plates only and the second experiment included 384-well plates). The calculation for the LLD was: 3SDBlank /slope, where SDBlank is the standard deviation of blank wells (media without cells) and the slope is the slope of the curve at the bottom end. (For the 96-well and 384-well plates respectively, the 10,000 cells/well and 2500 cells/well data points were used.) The RFU signal and standard deviation of wells containing non-transfected cells were similar to those containing media only.

The ZsGreen cell line had similar limits of detection in both experiments, whereas the other two differed as much as threefold between the experiments. We attribute these differences to the fact that their rates of growth, and therefore the number of passages, differed between cell lines and between the experiments. (With each successive passage, the cells become dimmer.) For example, in the first experiment, the DsRed cell line had 2-4 more passages than the other cell lines because it was growing more quickly. For better reproducibility, it would be better to design the experiments to keep the same passage number.


CONCLUSION
The Analyst GT multimode reader offers superior performance for measuring fluorescent proteins in intact adherent cells. Bottom reading gives better results, though top-read results are also quite good. The Analyst GT reader achieves its excellent performance with SmartOptics, a unique system that allows you to select and configure all components of the optical system to give you the best results for your assay. SmartOptics includes a speedy xenon flash lamp, as well as a powerful xenon continuous lamp so the correct light source can be used based upon assay mode. Also utilized in Analyst are high-quality fluorescence filters and fiber optics. Analyst has precise 3-axis positioning so that light is focused into individual wells and efficiently collected for maximum signal-to-background and sign al-to-noise ratios. The infinitely adjustable z-height, when reading from either the top or bottom of the microplate, allows you to focus directly on cells adhered to the well bottom or to read in the middle of a column of liquid in a homogeneous assay. Finally, the Analyst is compatible with several robotic systems, making it ideal for high-throughput screening systems.


REFERENCES
1. http://www.bdbiosciences.com/clontech


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