A high density assay format for the detection of novel cytotoxic agents in large chemical libraries

Figures & data

Figure 1.  (a) Growth curve of NCEB1 cell line with various cell densities as assessed by the AB reagent over a time course of 72 h after AB addition. Cells were seeded into 384-well microtiter plates. An average of 48 wells was used for each data point. (b) Effect of DMSO on the growth of NCEB1 cell line as assessed by the AB reagent over a time course of 48 h. Cells were seeded into 384-well microtiter plates. An average of 96 wells was used for each data point. (c) Assessment of assay performance and robustness. Cells were seeded into 384-well microtiter plates and incubated for 36 h prior to addition of the AB reagent. High control wells contained 1% DMSO (v/v). Low control wells contained 25 μM staurosporine in 1% DMSO (v/v).

Figure 2.  Effects of known cytotoxic agents on the growth of the NCEB1 cell line as assessed by the AB reagent over a time course of 36 h. Cells were seeded into 384-well microtiter plates and incubated for 36 h prior to the addition of the AB reagent. Negative control wells contained no cells. Positive control wells contained 1% DMSO (v/v). All drugs were screened at a 10 μM final concentration in 1% DMSO (v/v). An average of 48 wells was used for each data point.

Figure 3.  Assay miniaturization in 1536-well microtiter plate. Cells were seeded into 1536-well microtiter plates and incubated for 36 h prior to the addition of the AB reagent and further incubated for 24 h prior to reading the AB fluorescence. High control wells contained 1% DMSO (v/v). Low control wells contained 25 μM staurosporine in 1% DMSO (v/v).

Figure 4.  Control experiment to evaluate assay robustness and performance in 1536-well microtiter format. Top panel shows heat map analysis of the high and low control plate. Bottom panel shows AB signal and distribution of high and low control points. High control wells contained 1% DMSO (v/v). Low control wells contained 25 μM staurosporine in 1% DMSO (v/v).

Figure 5.  Sample images of 384-well microtiter plate and 1536-well microtiter plate as imaged by the LEADseeker™ Multimodality Imaging System.

Figure 6.  Scatter plot of 2000 compound validation of the NCEB1 cell line. Scatter plot shows percent inhibition of duplicate values for each compound.

Table I.  Comparison of IC₅₀ values in both 384-well microtiter plate and 1536-well microtiter plate. NCEB1 cells were seeded into 384-well microtiter plates and 1536-well microtiter plates and incubated with drugs for 48 h prior to addition of AB reagent. Cells were then incubated for another 24 h with AB reagent before readings were taken. High control wells contained 1% DMSO (v/v). Low control wells contained 25μM staurosporine in 1% DMSO (v/v).

			IC50(μm)
SKI ID	Structure	Compound name	384-Well	1536-Well
218440		Dihydrocelastryl diacetate	2.7	2.1
210404		Phenylmercuric acetate	2.8	2.2
209330		Aklavine hydrochloride	2.9	3.6
209418		Dihydrogambogic acid	3.8	2.3
218424		Pristimerin	3.8	2.8
210192		Oxyphenbutazone	4.0	2.0
210975		Cetrimonium Bromide	4.7	4.6
209302		Strophanthidinic acid lactone acetate	5.0	4.9
211301		Tomatine	5.0	4.2
209623		Acetyl isogambogic acid	5.4	4.2
218258		Pristimerol	5.6	4.2
210324		Thimerosal	6.6	4.3
210622		Peruvoside	0.1	0.1
210432		Ouabain	0.2	0.1
209981		Pyrithione zinc	0.4	0.4
209966		Digoxin	0.8	0.6
209298		Cymarin	1.0	0.6
210042		Gentian violet	1.2	1.2
218346		Oleandrin	1.3	1.2
210614		Puromycin hydrochloride	1.5	1.3
211052		Mitoxanthrone hydrochlohide	1.5	1.9
218427		Dimethyl gambogate	1.7	1.5
209297		Digoxigenin	1.8	1.8
210193		Oxyquinoline	2.0	1.8
218300		Rutilantinone	6.7	6.7
209965		Digitoxin	6.7	5.1
211104		Benialkonium chloride	7.7	7.3
211166		Homidium bromide	8.0	7.0
209537		3,4-Dimethoxydalbergione	8.3	5.9
218245		Cytochalasin A	8.5	5.8
209735		Propachlor	8.5	5.8
211332		Rhodomyrtoxin B	9.4	8.4
211032		Methylbenzethonium chloride	10.6	8.9
209881		Chlorhexidine	11.1	9.0
209907		Clomiphene citrate	24.5	19.8
211495		Amoxepine	72.7	63.1

Figure 7.  (a) IC₅₀ curves of each compound in 384-well microplate format: Digoxin, 0.8 μM; Cytochalasin A, 8.5 μM; and Clomiphene Citrate, 24.5 μM. (b) IC₅₀ curves of each compound in 1536-well microplate format: Digoxin, 0.6 μM; Cytochalasin A, 5.8 μM; and Clomiphene Citrate, 19.8 μM. (c) Correlation plot of IC₅₀ values in 384-well microtiter plate versus 1536-well microtiter plate. NCEB1 cells were incubated with compounds for 48 h and then incubated with AB reagent for another 24 h. High control wells contained 1% DMSO (v/v). Low control wells contained 25 μM staurosporine in 1% DMSO (v/v). IC₅₀ values are an average of two independently fitted dose response curves for each data set.