P75ntr Screening Assay

摘要

The present invention provides a method to identify a test compounds capability to modulate p75^NTR induced apoptosis, said method comprising: i. Transfecting a suspension of eukaryotic cells with a vector encoding p75^NTR (SEQ ID No.2) or a cell death inducing fragment thereof, ii. Contacting said cells with the compound to be tested, and iii. Determine the apoptotic response in said cells, wherein an alteration in apoptotic response in the presence of said test compound compared to the apoptotic response in the absence of the test compound is an indication of the ability of the test compound to modulate p75^NTR induced apoptosis. In this method according to the invention the suspension of eukaryotic cells consist of Hek293T cells used at a cell density of 0.4-3.0x10⁴ cells/100 mul and transfected in the presence of a lipid-based transfection reagent at ratio of transfection reagent to DNA of 6-1, in particular at a ratio of 4. Expressed per 10 ml of final transfection mix the amount of transfection reagent is in range of 8.0-12.0 mul and the amount of DNA is in a range of 2.0-3.5 mug. The apoptotic response of the cells in the method according to the invention is determined using art known procedures. In particular using annexin V or nuclear staining. In a preferred embodiment the apoptotic response is determined using Annexin-V-Alexa Fluor 488 and Hoechst 33342. The p75^NTR cell death inducing fragment as used hereinbefore comprises the p75 Chopper domain (SEQ ID No.10) and in particular consists of p75_ICD (SEQ ID No.4), p75_CD (SEQ ID No.6) or p75_TNF (SEQ ID No.8).