MICROPLATE EDGE EFFECTS IN RANGE FINDING STUDIES USING THE
LUMI-CELL  ER BIOASSAY: INCREASING ASSAY THROUGHPU TGordon, J.D.1 , Denison, M.S. 2, Chu, A.C.1, Chu, M.D.3, Matherly, C.1, and Clark, G.C.1
1
Xenobiotic Detection Systems Inc., 1601 E. Geer St. Suit e S, Durham, NC 27704 Phone: (919) 688-4804;
FAX: (919) 688-4404; 2Department of Environmental Toxicology, Meyer Hall, Univ. of California, Davis, CA95616. 3Analytical Perspectives, 2714 Exchange Drive, Wilmington, NC 28405.
Abstract
Concern over the detrimental effects of endocrine disruptor chemicals (EDCs) has prompted the need for aneffective high throughput system for detection of EDCs. Typically, the outer wells of a 96-well plate are notused for bioassay analysis due to edge effects, which have been found to reduce measured activity. This resultsin fewer available assay wells, in effect decreasing analysis throughput by 38%. Ring analysis of the 96-wellplate and range finding studies with Bisphenol A and Bisphenol B in the LUMI-CELL ER bioassay revealed
no significant difference (after t-test analysis) between using the inner 60 or all 96 wells of a 96-well microplate.
Utilizing all 96 wells in the assay will increase throughput of the assay by 38% over that of typical cellbioassays that use only the 60 inner wells of the plate.
Introduction
The strong association between the exposure and bioaccumulation of endocrine disruptor chemicals (EDCs) andtheir adverse effects on human and wild life populations has given rise to concerns worldwide over the past
several years1,2 . Some examples of the effects of EDCs are: decreased reproductive success and feminization ofmales in several wildlife species; increased hypospadias along with reductions in sperm counts in men; increasein the incidence of human breast and prostate cancers; and endometriosis 3- 5. In light of these detrimental effects
of environmental exposure to EDCs, there is an obvious need to increase throughput of screening systems fordetection of EDCs.
In typical microplate cell bioassays, the outer wells of a 96-well plate are not used because of “edge” effects.
These edge effects were indicated to result in decreased sensitivity and increased variability in sample readings
as a result of a lack of constant humidity and temperature resulting in uneven evaporation7. While there areproducts produced, such as covered plates and incubators with faster temperature and humidity recovery times,
to reduce edge effects, there are very few published studies, which actually document edging effects. Lack ofinclusion of the 36 outer wells of a 96-well plate in bioassays, results in a reduction in sample throughput,
effectively reducing assay throughput by 38% of total capacity. In this report we conducted 2 studies toexamine the variability of the “outer” wells compared to that of the “inner” 60 wells. Bisphenol A (BPA) andBisphenol B (BPB) from the ICCVAM sponsored protocol standardization study of the LUMI-CELL ER
bioassay were used to determine variability in actual range finding studies using both inner and outer wells.
Methods and Materials
Compound Preparation and Analysis Procedure: In the first study a dosing concentration of 2.5x10-5 µg/ml
17β-estradiol (E2) or 3.13 µg/ml Methoxychlor was plated in all 96 wells. An analysis of the outer ring and all
successive inner rings were then compared to the inner 60 wells. The data from two plates of E2 and one plateof Methoxychlor were averaged and the data presented here. In the second study, a 10 mg/ml solution of eachcompound (BPA and BPB) was prepared in DMSO. A range finding assay was preformed on each compoundusing six log dilutions. A group comparison t-Test was preformed to determine any significant differences.
LUMI-CELL ER Bioassay. The BG1Luc4E2 cell line was constructed as previously described by Rogers andDenison (2000). Briefly, BG1 cells were stably transfected with an estrogen-responsive luciferase reporter gene
plasmid (pGudLuc7ere) and selected for G418 resistance5 . For analysis, the BG1Luc4E2 cell clone resulting
from this selection was grown in RPMI 1640 medium. The cells were transferred into flasks containing phenol
red-free DMEM media supplemented with charcoal dextran stripped FBS and incubated for four days before
harvesting for BG1Luc4E 2 bioassay plating. The cells were then plated in white clear -bottomed Corning 96 wellmicroplates and incubated at 37oC for 24-48 hours prior to dosing. The media solution in each well wasremoved and two hundred microliters of phenol red-free DMEM supplemented with charcoal dextran stripped
FBS containing the indicated concentration of the desired chemical to be tested was added to each well. Theplate was then incubated for 20 hours 37oC before analysis of luciferase activity.
Measurement of Luciferease Activity. After lysing the cells (Promega lysis buffer), the luciferase activity wasmeasured in a Berthold Orion Microplate Luminometer, with automatic injection of 50 micro liters of luciferase
enzyme reagent (Promega) into each well. The relative light units (RLUs) measured were compared to thatinduced by the E2 standard after subtraction of the background activity.
Results and Discussion
In the first study, a ring analysis was conducted of the 96-well plates. The entire plate was dosed with one
concentration of E2 (2.5x10- 5 µg/ml (91.8 pM)) or Methoxychlor (3.13 µg/ml (905.4 µM)). The data from twoplates of E2 and one plate of Methoxychlor were averaged and the data presented here. The outer ring (Ring 1)
and each successive inner ring were compared to the normally used inner 60 wells (Figure 1). Luciferase
activity, of lysed cells in wells in Ring 1, was 7% lower than that in the inner 60 wells, while activity in celllysates in wells in Ring 2 were 3% lower. Cell lysates in wells in Rings 3 and 4 were each 1% higher than thatof lysates in the inner 60 wells. Each of the top and bottom rows, as well as the left and right columns were also
compared to the inner 60 wells. The outer wells were compared by row and column (Table 1B) and ranged
from 4% - 10% lower than the inner 60 wells. None of the ring analysis or any of the outer rows and columns
demonstrated any significant difference from the inner 60 wells when analyzed by t-test (Tables 1A and 1B).
While our results demonstrate an overall decrease in activity in cell lysates in the outer wells, the activities weremuch higher and less variable than previously reported, indicating the potential of using these wells for LUMICELL ER screening or range finding studies.
Figure 1. Ring analysis of 96-well plate using the LUMI-CELL ER bioassay. Each ring of data (Ring 1, 2, 3,and 4) was compared to the inner 60 wells contained within the dashed lines below.
123456789101112

Ring 1
Ring 2ABC

Ring 3
Ring 4

DEFGH

Table 1A and 1B. Ring and outer well analysis of RLU Data from E2 and Methoxychlor on 96 -well plate. In
Table 1A, the mean from each ring was compared to the mean of the inner 60 wells. In Table 1B, mean fromeach outer row and column was compared to the mean of the inner 60 wells.
AB

Ring 1
Ring 2
Ring 3
Ring 4

Mean
93%
97%
101%
101%

SD
5%
1%
1%
1%

t0.95
0.92
1.04
-0.63
-0.49

top rowbottom row
lef column
right column

Mean
94%
90%
91%
96%

SD
7%
6%
3%
10%

t0.95
0.53
0.84
1.16
0.55

In the second study, a range finding study was conducted to determine the edge effects on actual samples.
Solutions of BPA and BPB (10 mg/ml each) were prepared in DMSO. A range finding assay was preformed oneach compound using seven log dilutions. Each compound was then run vertically in the 96 -well plate using theouter wells in triplicate in two locations on the plate (Figure 2A). These same compounds were then run
horizontally within the normal inner 60 wells used in most assays (Figure 2B). The results from each plate werecompared to determine the variability of the two dosing methods. For both BPA and BPB, there was nosignificant difference (t 0.95 ≤ 2.09 for all concentrations of BPA and t0.95 ≤ 3.16 for all concentrations of BPB) inthe response of the LUMI-CELL  ER bioassay when the compounds were plated vertically using the outer wells
and dosed horizontally using just the inner 60 wells (Figure 3 and 4). The far left column in Figure 2A, testingBPA, demonstrates no significant difference (t0.95 ≤ 1.00 for all concentrations tested) from any of the otherinner columns dosed. The far right column in Figure 2A, testing BPB, demonstrates no significant difference(t 0.95 ≤ 2.08 for all concentrations tested) from any of the other analysis. There appears to be no significant
difference in the data from the current study and the data submitted to ICCVAM for the LUMI-CELL ERProtocol Standardization Study, which was dosed as in Figure 2B and seen in Figures 3 and 4. The highest
concentration for both BPA and BPB were cytotoxic and therefore not included in the graphs.
Figure 2. Range finder analysis for BPA and BPB using the LUMI-CELL ER bioassay. Figure 2A depicts theplate set up for analysis using all 96 wells. Figure 2B depicts the plate setup for normal test ing using only theinner 60 wells.
AB- Bisphenol A
1234- Bisphenol B
567891011123456789101112A

12A

BBCCDDEEFFGGHH

E2- E2 Standard Dose Response Curve- Methoxychlor (313 µg/ml) (positive control)- DMSO (solvent/negative control)

DMSO
E2Methoxychlor- Bisphenol A- Bisphenol B

Figure 3. Range finder analysis of BPA analyzed by both plate setups depicted in Figure 2A (normal) and 2B(96). The BPA - ILS line represents the data for BPA presented to ICCVAM during the protocol
standardization study for the LUMI-CELL ER bioassay.
Bisphenol A - Normal and all 96 wells
DMSO

Methoxychlor Control

Avg Adj RLU

BPA (a)- normal

BPA (b)- normal
BPA c-96

BPA (c)- normal
BPA d-96

BPA a-96
BPA e-96

PBA b-96
BPA f-96

16000 E2 Bottom

BPA - ILS

adj. RLU above background

14000
12000
10000
8000
6000
4000
2000
0-20001.00.E-08

1.00.E-06

1.00.E-04

1.00.E-02µg/ml

1.00.E+00

1.00.E+02

1.00.E+04

Figure 4. Range finder analysis of BPB analyzed by both plate setups depicted in Figure 2A (normal) and 2B(96). The BPB - ILS represents the data for BPB presented to ICCVAM during the protocol standardization
study for the LUMI-CELL ER bioassay.
Bisphenol B - Normal and all 96 wells
DMSO

Methoxychlor Control

Avg Adj RLU

BPB (a)- normal

BPB (b)- normal
BPB d-96

BPB a-96
BPB e-96

BPB b-96
BPB f-96

BPB c-96
E2 Bottom

BPB - ILS

16000

adj. RLU above background

14000
12000
10000
8000
6000
4000
2000
0-20001.00.E-08

1.00.E-06

1.00.E-04

1.00.E-02

1.00.E+00

1.00.E+02

1.00.E+04µ g/mlThe data presented here using the ring analysis and range -finder analysis, demonstrate the responsiveness andlow variability of the outer wells of a 96-well plat e in the LUMI-CELL ER bioassay. After t -test analysis of alldata there was no significant difference in any of the data analysis. This data further supports the use of thesewells in screening or range finding studies, which will result in a 38% increase in sample throughput for theLUMI-CELL ER bioassay. Studies are currently in progress to determine whether similar results are obtainedwith other cell based bioassays (i.e. XDS-CALUX bioassays for dioxin-like chemicals and androgenicchemicals).
Acknowledgements: This work was supported in part by grants from the National Institutes of Environmental
Health Small Business Initiated Research program (ES10533 -03) and a Superfund Basic Research Grant(ES04699).
References
1. Jarry H., Christoffel J., Rimoldi G., Koch L., Wuttke W. (2004). Toxicology. 205(1-2):87-93.
2. Jefferson W.N., Padilla-Banks E., Clark G., Newbold R.R. (2002). J Chromatogr B Analyt Technol
Biomed Life Sci. 777:179-189.
3. Markey C.M., Coombs M.A., Sonnenschein C., Soto A.M. (2003). Evol Dev. 5:67-75.
4. Safe S.H. (2002). Health Perspect. 110: 925-929.
5. Rogers J.M, and Denison M.S. (2000). In Vitro Mol Toxicol. 13:67-82.
6. Sonneveld E., Riteco J., Jansen H., Pieterse B., Brouwer A., Schoonen W., Burg B. (2005). Toxicol Sci.
89:173-187.
7. de Johgh, G. (1999). http://www.bio.net/bionet/mm/methods/1999-May/075278.html
8. Nomination of the LUMI-CELL ER bioassay High Throughput System for Screening Estrogen-Like
Chemicals for Validation Studies . http://iccvam.niehs.nih.gov/methods/endocrine/endodocs/XDSeval2.pdf
9. Current Evaluation of In Vitro Test Methods for Detecting Potential Endocrine Disruptors (May 2003).
ICCVAM, NTP, NICEATM, NIEHS. NIH Publication No. 03-4503.
10. Current Status of Test Methods for Detecting Endocrine Disruptors: In Vitro Estrogen Receptor
Transcriptional Activation. http://iccvam.niehs.nih.gov/docs/docs.htm#endocrine andhttp://iccvam.niehs.nih.gov/methods/endodocs/final/erta_brd/erta_all.pdf.