By Hao Zhu, Menghang Xia
This ebook specializes in lately constructed excessive Throughput Screening (HTS) assay protocols, many fascinated about the ToxCast and/or Tox21 tasks, and the appropriate HTS information research strategies. Divided into 3 sections, in vitro assays, in vivo assays, and computational suggestions to research HTS info are all tested. Written for the hugely winning Methods in Molecular Biology sequence, so much chapters contain introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, conveniently reproducible laboratory protocols, and tips about troubleshooting and averting identified pitfalls.
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Am J Phys 271(4 Pt 1):C1172–C1180 3. Wang GL, Semenza GL (1995) Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 270(3):1230–1237 4. Huang LE, Gu J, Schau M, Bunn HF (1998) Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A 95(14):7987–7992 5. Salceda S, Caro J (1997) Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions.
2. Fit concentration–response curves of normalized data points to determine IC50 and efficacy values using a four-parameter Hill equation in GraphPad Prism software (Figs. 1, 2, 3, and 4). Fig. 1 Concentration–response curves of cobalt chloride in HRE-bla normoxia mode assay. Each data point is presented as mean ± SD from duplicates Fig. 2 Concentration–response curves of topotecan in HRE-bla hypoxia mode assay. Each data point is presented as mean ± SD from duplicates Fig. 3 Concentration–response curves of cobalt chloride in HIF-1α-NanoLuc normoxia mode assay.
Check confluency of the cells; they should be about 70–90 % confluent to proceed to the next step. 5. Use Table 2 to combine the proper amounts of each reagent to the wells of plated HepG2 cells. Let the mix sit for 5–25 min at room temperature before adding to the well. 6. 75 mL of transfection medium. 7. Add 250 μL of the transfection mix into each well and incubate at 37 °C and 5 % CO2 overnight. 8. Trypsinize two of the wells and reseed cells into about ten 10 cm2 dishes using the culture medium for HepG2-CYP2B6hCAR cells for selection.