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Optimization of cell-based assays to quantify the anti-inflammatory/allergic potential of test substances in 96-well format

Inflammopharmacology volume 19, pages 169–181 (2011)Cite this article

Abstract

Objective

There is an insistent need for robust, reliable, and optimized assays for screening novel drugs targeting the inflammatory/allergic markers. The present study describes about the optimization of eight cell-based assays utilizing mammalian cell lines in 96-well format for quantifying anti-inflammatory/allergic drug candidates.

Materials and methods

We estimated the inhibitory response of reference compounds: 1400W dihydrochloride on LPS-induced NO release, celecoxib on LPS-induced PGE2 production and dexamethasone on LPS-induced pro-inflammatory cytokines IL-1 beta, IL-6, and TNF-alpha production by J774A.1 murine macrophages. Response of acetylsalicylic acid and celecoxib was studied on A23187-induced TXB2 production; captopril on A23187-stimulated LTB4 production by HL-60 cells. Effect of ketotifen fumarate was evaluated on A23187-elicited histamine release by RBL-2H3 cells. Each experiment was repeated twice to assess the reproducibility and suitability of the assays by determining appropriate statistical tools viz. %CV, S/B and Z′ factor.

Results

1400W dihydrochloride was capable of inhibiting LPS-induced NO levels (IC50 = 10.7 μM). Dexamethasone attenuated LPS-induced IL-1 beta (IC50 = 70 nM), IL-6 (IC50 = 58 nM) and TNF-alpha (IC50 = 44 nM) release, whereas celecoxib, a specific COX-2 inhibitor showed marked reduction in LPS-induced PGE2 (IC50 = 23 nM) production. Captopril (IC50 = 48 μM) and ketotifen fumarate (IC50 = 36.4 μM) demonstrated potent inhibitory effect against A23187-stimulated LTB4 and histamine levels, respectively. Both acetylsalicylic acid (IC50 = 5.5 μM) and celecoxib (IC50 = 7.9 nM) exhibited concentration-dependent decrease in TXB2 production. Results for all the cell assays from two experiments showed a Z′ factor varying from 0.30 to 0.99; the S/B ratio ranged from 2.39 to 24.92; %CV ranged between 1.52 and 20.14.

Conclusion

The results proclaim that these cell-based assays can act as ideal tools for screening new anti-inflammatory/anti-allergic compounds.

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Abbreviations

NO:

Nitric oxide

PGE2 :

Prostaglandin E2

IL-1 beta:

Interleukin-1 beta

IL-6:

Interleukin-6

TNF-alpha:

Tumor necrosis factor-alpha

LTB4 :

Leukotriene B4

TXB2 :

Thromboxane B2

LPS:

Lipopolysaccharide

A23187:

Calcimycin

%CV:

Coefficient of variation

S/B :

Signal to background ratio

SD:

Standard deviation

IC50 :

Half maximal inhibitory concentration

ATCC:

American type culture collection

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Acknowledgments

We thank Mr. P. Thiyagarajan and Mr. H.B. Deepak, Department of Cellular Assay, for their technical assistance. The authors are grateful to Mr. Prashanth D’Souza, R&D Centre, Natural Remedies Pvt. Ltd., Bangalore, India, for providing technical details about assay performance measures.

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  1. Department of Cellular Assay, R&D Centre, Natural Remedies Pvt. Ltd, Plot No. 5B, Veerasandra Industrial Area, 19th K. M. Stone, Hosur Road, Bangalore, 560100, India

    C. V. Chandrasekaran, R. Edwin Jothie, Preeti Kapoor, Anumita Gupta & Amit Agarwal

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  1. C. V. Chandrasekaran
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  2. R. Edwin Jothie
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Correspondence to C. V. Chandrasekaran.

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Chandrasekaran, C.V., Edwin Jothie, R., Kapoor, P. et al. Optimization of cell-based assays to quantify the anti-inflammatory/allergic potential of test substances in 96-well format. Inflammopharmacol 19, 169–181 (2011). https://doi.org/10.1007/s10787-010-0065-1

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Keywords

  • 1400W dihydrochloride
  • Dexamethasone
  • Celecoxib
  • Captopril
  • Acetylsalicylic acid
  • Ketotifen fumarate
  • Inflammatory mediators
  • Z′ factor
  • S/B
  • %CV