Hello, I am working on cell culture and wish to perform inflammatory assays, especially, COX and LOX. I have found several kits of different companies. Due to financial limitations, I think its better to adopt some methods without using kits. Can anyone please help me?
Dear Abhilash,
The following publication covers the answer to your question.
Indian J Biochem Biophys. 2011 Aug;48(4):256-61.
High-throughput screening assays for cyclooxygenase-2 and 5-lipoxygenase, the targets for inflammatory disorders.
Kumar KA1, Reddy TC, Reddy GV, Reddy DB, Mahipal SV, Sinha S, Gaikwad AN, Reddanna P.
http://www.ncbi.nlm.nih.gov/pubmed/22053694
Abstract
High-throughput screening (HTS) involves testing of compound libraries against validated drug targets using quantitative bioassays to identify 'hit' molecules that modulate the activity of target, which forms the starting point of a drug discovery effort. Eicosanoids formed via cyclooxygenase (COX) and lipoxygenase (LOX) pathways are major players in various inflammatory disorders. As the conventional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit both the constitutive (COX-1) and the inducible (COX-2) isoforms have gastric and renal side effects and the recently developed COX-2 selective anti-inflammatory drugs (COXIBs) have cardiac side effects, efforts are being made to develop more potent and safer antiinflammatory drugs. Current assay methods for these enzymes, such as oxygraphic, radioisotopic, spectrophotometric etc. are not compatible for screening of large number of compounds as in drug discovery programs. In the present study, HTS-compatible assays for COX-1, COX-2 and 5-LOX were developed for screening of compound libraries with the view to identify potential anti-inflammatory drug candidates. A spectrophotometric assay involving co-oxidation of tetramethyl-p-phenylene diamine (TMPD) during the reduction of prostaglandin G2 (PGG2) to PGH2 was adopted and standardized for screening of compounds against COX-1 and COX-2. Similarly, the HTS-compatible FOX (ferrous oxidation-xylenol orange) based spectrophotometric assay involving the formation of Fe3+/xylenol orange complex showing absorption in the visible range was developed for screening of compounds against 5-LOX.
Hoping this will be helpful,
Rafik
Dear Abhilash,
The following publication covers the answer to your question.
Indian J Biochem Biophys. 2011 Aug;48(4):256-61.
High-throughput screening assays for cyclooxygenase-2 and 5-lipoxygenase, the targets for inflammatory disorders.
Kumar KA1, Reddy TC, Reddy GV, Reddy DB, Mahipal SV, Sinha S, Gaikwad AN, Reddanna P.
http://www.ncbi.nlm.nih.gov/pubmed/22053694
Abstract
High-throughput screening (HTS) involves testing of compound libraries against validated drug targets using quantitative bioassays to identify 'hit' molecules that modulate the activity of target, which forms the starting point of a drug discovery effort. Eicosanoids formed via cyclooxygenase (COX) and lipoxygenase (LOX) pathways are major players in various inflammatory disorders. As the conventional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit both the constitutive (COX-1) and the inducible (COX-2) isoforms have gastric and renal side effects and the recently developed COX-2 selective anti-inflammatory drugs (COXIBs) have cardiac side effects, efforts are being made to develop more potent and safer antiinflammatory drugs. Current assay methods for these enzymes, such as oxygraphic, radioisotopic, spectrophotometric etc. are not compatible for screening of large number of compounds as in drug discovery programs. In the present study, HTS-compatible assays for COX-1, COX-2 and 5-LOX were developed for screening of compound libraries with the view to identify potential anti-inflammatory drug candidates. A spectrophotometric assay involving co-oxidation of tetramethyl-p-phenylene diamine (TMPD) during the reduction of prostaglandin G2 (PGG2) to PGH2 was adopted and standardized for screening of compounds against COX-1 and COX-2. Similarly, the HTS-compatible FOX (ferrous oxidation-xylenol orange) based spectrophotometric assay involving the formation of Fe3+/xylenol orange complex showing absorption in the visible range was developed for screening of compounds against 5-LOX.
Hoping this will be helpful,
Rafik
Another vote for TMPD, this is the most straightforward way to do it:
http://www.ncbi.nlm.nih.gov/pubmed/20072914
5 g for $53, 25 g for $175 in the US as of Dec 2015, hope it's not too different in India:
http://www.sigmaaldrich.com/catalog/product/sial/87890?lang=en®ion=US
I perform western blot for COX-2 expression but will have a look at the protocol Rafik is suggesting here
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