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Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells.

Leister KP, Huang R, Goodwin BL, Chen A, Austin CP, Xia M - Curr Chem Genomics (2011)

Bottom Line: These compounds were also confirmed in a traditional ELISA assay.We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316.The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.

View Article: PubMed Central - PubMed

Affiliation: NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.

No MeSH data available.


Related in: MedlinePlus

(A) Time course of LPS-induced TNF-α production. THP-1 cells were treated with various LPS concentrations for 5, 17 and 24 hr. At the end of various time points, TNF-α production was measured in THP-1 cells using a homogenous HTRF-based TNF-α assay. Data are from a single experiment performed in quadruplicate, representative of several experiments. (B) Optimization of cell density. THP-1 cells were dispensed at 2k, 3k and 4k per well. After incubated with LPS for 17 hr, TNF-α production in the cells was measured. Data are from a single experiment performed in quadruplicate.
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Figure 4: (A) Time course of LPS-induced TNF-α production. THP-1 cells were treated with various LPS concentrations for 5, 17 and 24 hr. At the end of various time points, TNF-α production was measured in THP-1 cells using a homogenous HTRF-based TNF-α assay. Data are from a single experiment performed in quadruplicate, representative of several experiments. (B) Optimization of cell density. THP-1 cells were dispensed at 2k, 3k and 4k per well. After incubated with LPS for 17 hr, TNF-α production in the cells was measured. Data are from a single experiment performed in quadruplicate.

Mentions: We have optimized and validated a homogenous HTRF-based TNF-α assay in a 1536-well plate format that can be used to screen compounds to identify potential TNF-α inhibitors (Fig. 1). LPS, a known TNF-α stimulator, induced TNF-α production in a concentration-dependent manner after 17 hr incubation with the THP-1 cells (Fig. 4A). The EC50 of LPS was 0.84 µg/ml, and the maximum induction of TNF-α production by LPS was more than 8-fold of the basal level.


Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells.

Leister KP, Huang R, Goodwin BL, Chen A, Austin CP, Xia M - Curr Chem Genomics (2011)

(A) Time course of LPS-induced TNF-α production. THP-1 cells were treated with various LPS concentrations for 5, 17 and 24 hr. At the end of various time points, TNF-α production was measured in THP-1 cells using a homogenous HTRF-based TNF-α assay. Data are from a single experiment performed in quadruplicate, representative of several experiments. (B) Optimization of cell density. THP-1 cells were dispensed at 2k, 3k and 4k per well. After incubated with LPS for 17 hr, TNF-α production in the cells was measured. Data are from a single experiment performed in quadruplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3106354&req=5

Figure 4: (A) Time course of LPS-induced TNF-α production. THP-1 cells were treated with various LPS concentrations for 5, 17 and 24 hr. At the end of various time points, TNF-α production was measured in THP-1 cells using a homogenous HTRF-based TNF-α assay. Data are from a single experiment performed in quadruplicate, representative of several experiments. (B) Optimization of cell density. THP-1 cells were dispensed at 2k, 3k and 4k per well. After incubated with LPS for 17 hr, TNF-α production in the cells was measured. Data are from a single experiment performed in quadruplicate.
Mentions: We have optimized and validated a homogenous HTRF-based TNF-α assay in a 1536-well plate format that can be used to screen compounds to identify potential TNF-α inhibitors (Fig. 1). LPS, a known TNF-α stimulator, induced TNF-α production in a concentration-dependent manner after 17 hr incubation with the THP-1 cells (Fig. 4A). The EC50 of LPS was 0.84 µg/ml, and the maximum induction of TNF-α production by LPS was more than 8-fold of the basal level.

Bottom Line: These compounds were also confirmed in a traditional ELISA assay.We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316.The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.

View Article: PubMed Central - PubMed

Affiliation: NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.

No MeSH data available.


Related in: MedlinePlus