Method for detecting expression level of annexin A3
A technology for annexin and tumor cells, applied in the field of detecting the expression level of annexin A3
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Embodiment 1
[0055] Example 1: In vitro study on the mechanism of annexin A3 regulating the drug resistance of ovarian cancer epithelial cells to platinum-based chemotherapy drugs
[0056] 1. Experimental method
[0057] 1) Cell lines and their culture conditions
[0058] Human epithelial ovarian cancer cell SKOV3 cell line was purchased from the Basic Medical Cell Center of the Chinese Academy of Medical Sciences. SKOV3 / Cis is the SKOV3 cisplatin-resistant cell subline induced by the inventor (refer to Yan Xuedong, Zhang Mingwei, Pan Lingya. Basic Medicine and Clinical, 2006, 26(7):739-744). This experiment also used A2780 and its cisplatin-resistant cell line A2780 / Cis (refer to LuanYZ, Li L. et al. Zhonghua Fu Chan Ke Za Zhi, 2004, 39(6): 403-407.). These cells were all transfected with an empty pcDNA3.1(+) plasmid. SKOV3 / Ann and A2780 / Ann cell lines are stable transfection clones obtained after SKOV3 and A2780 were transfected with positive-sense Annexin A3 plasmids, respectively, a...
Embodiment 2
[0083] Example 2: Identification of Exocrine Annexin A3 in Epithelial Ovarian Cancer Cells
[0084] 1. Experimental method
[0085] 1) Cell lines and cell culture
[0086] With embodiment 1.
[0087] 2) Collection and concentration of epithelial ovarian cancer cell line culture supernatant
[0088] Take 2×10 5 Cells were seeded at 25cm 2 Cell culture flasks adhered to the wall and replaced serum-free DMEM when the growth reached 70%, about every 1×10 6 The cells were given 5ml of culture medium, and placed in a cell incubator to continue culturing for 48 hours. Then, the serum-free DMEM was collected in a 10ml centrifuge tube, and centrifuged at 1000g for 10 minutes to remove the formed components in DMEM. After centrifugation, the supernatant was carefully collected and placed Centrifuge horizontally at 3000g for 15 minutes in an Amicon Ultra-15 ultrafiltration tube (Millipore, USA), collect the concentrated liquid in an Ep tube, and store at -70°C.
[0089] 3) Semi-qua...
Embodiment 3
[0104] Example 3: The Exocrine Pathway of Annexin A3 in Ovarian Cancer Cells
[0105] 1. Experimental method
[0106] 1) Cell lines and culture
[0107] The cells used in this experiment and their culture conditions are completely consistent with those in "Example 2", and will not be repeated here.
[0108] 2) Immunofluorescence and its laser confocal imaging
[0109] Take 2×10 4 Cells were planted in a 24-well cell culture plate with glass slides, at 37°C, 5% CO2 After culturing overnight in a saturated humidity incubator, discard the medium, wash 3 times with PBS, fix with 4% paraformaldehyde for 20 minutes, wash 3 times with PBS, permeabilize the membrane with 0.5% TritonX-100 for 20 minutes, wash 3 times with PBS, 2% Blocked with BSA for 30 minutes, incubated with 1:50 rabbit anti-human annexin A3 polyclonal antibody (primary antibody), overnight at 4°C. Wash 3 times with PBS, incubate with goat anti-rabbit FITC secondary antibody for 1 hour, wash 3 times with PBS, inc...
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