Application of PI3K/Akt signal path to Marek's disease virus multiplication in chicken embryo fibroblast and detection method of PI3K/Akt signal path
A technology of fibroblasts and signaling pathways, applied in the field of Marek's virus proliferation, achieving good repeatability, strong sensitivity, and optimized PCR reaction system and conditions
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0048] Example 1 Cytotoxicity Detection
[0049] This example detects the effects of different concentrations of PI3K-specific inhibitor LY294002 on the toxicity of chicken embryo fibroblasts, and the specific steps are as follows:
[0050] (1), using DMEM medium to prepare the PI3K specific inhibitor LY294002 to different concentrations of 5 μmol / L, 10 μmol / L, 20 μmol / L, 30 μmol / L, and 50 μmol / L;
[0051] (2) Simultaneously inoculate 100 μL / well chicken embryo fibroblast suspension in a 96-well plate. After the cells cover a single layer, discard the culture medium, wash twice with PBS, and add new DMEM containing 2% FBS for culture. Subsequently, the inhibitors of different concentrations in the above step (1) were added to a 96-well plate, 10 μL per well, and each concentration was repeated for 8 wells, and an equal amount of 0.1% DMSO was added as a control;
[0052] (3) Incubate the well plate after adding the inhibitor in a 5% CO2, 37°C incubator for 24 hours; add 10 μL...
Embodiment 2
[0054] Example 2 Western blot detection
[0055] (1), protein extraction: chicken fibroblasts are cultured into monolayer cells with DMEM nutrient solution containing 10% newborn calf serum in a sterile culture dish of 35 mm × 10 mm, and then add 20 μmol / L inhibitor LY294002;
[0056] Subsequently, inoculate 2×10 5 The Md5 of the PFU virus amount, collected samples of different time periods (0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 60h) after inoculation, and pre-conditioned at 4°C Wash twice with cold PBS, aspirate the PBS washing solution, add 200 μL RIPA protein lysate containing 5% protease inhibitor (PMSF), place on ice for 10 minutes, scrape the cells with a cell scraper, aspirate into a new 1.5mL centrifuge In the tube, beat several times with a 1mL syringe, centrifuge at 12000rpm at 4°C for 10min, take the supernatant, add 5×SDS-PAGE loading buffer, boil the sample in boiling water for 5min to denature the protein, and extract the total protein of chicken fibrobl...
Embodiment 3
[0060] Example 3 Virus Titration Determination
[0061] Inoculate chicken fibroblasts into two 96-well plates, discard the liquid after the monolayer is overgrown, wash twice with PBS, add new DMEM culture medium containing 2% FBS, first add LY294002 to each well of one of the well plates to inhibit The virus solution was incubated for 1 h, and then the virus solution was serially diluted 2 times with DMEM culture solution containing 2% FBS, and each dilution was inoculated into 8 wells, and at the same time, 2 columns of the other well plate were not inoculated with the virus as a blank control. 37°C, 5% CO 2 After culturing, PFU counting was carried out at 24h, 48h, 72h, and 96h respectively, and the results were as follows: image 3 shown.
[0062] Depend on image 3 It can be seen that, compared with the cells without LY294002 inhibitor, the virus titer in fibroblasts of chickens treated with LY294002 inhibitor was significantly reduced.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More - R&D
- Intellectual Property
- Life Sciences
- Materials
- Tech Scout
- Unparalleled Data Quality
- Higher Quality Content
- 60% Fewer Hallucinations
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com