Non-tumorigenic mdck cell line and screening method for amplifying influenza virus
An influenza virus, non-tumorigenic technology, applied in microorganism-based methods, animal cells, viruses/phages, etc., can solve the problems of non-tumorigenicity, 1-100 cells may form tumors, etc., and achieve cost savings , the effect of shortening the time
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Embodiment 1
[0051] Embodiment 1, have the acquisition of non-tumorigenic MDCK-2B2 cell line
[0052] 1. Obtaining MDCK monoclonal cells
[0053] 1. Cell digestion and counting
[0054] Passage ATCC CCL-34 MDCK P55 in serum-containing medium (Minimum essential medium + 10% fetal bovine serum (FBS) + 2mM glutamine) for a limited number of passages, MDCK P63 generation cells, select a bottle of T-25 cells for Digestion and counting.
[0055] Take a bottle of MDCK P63 T-25 cells, discard the culture supernatant, wash twice with 1mL trypsin (purchased from Gibco), then add 1mL trypsin, put in CO 2 Incubate in an incubator, observe under the microscope that the cells become round after 15 minutes, add 9 mL of medium to stop digestion, mix the cell suspension, take 10 μL and drop it on a cell counting plate for counting.
[0056] 2. Cell density adjustment and plating
[0057]In order to ensure that there is no single-well polyclonal existence in the 96-well cell plate, reduce the total numb...
Embodiment 2
[0067] Growth morphology and biochemical indicators of embodiment 2, MDCK-2B2 cells
[0068] 1. MDCK-2B2 cells have epithelial-like morphology
[0069] P85 MDCK-2B2 was observed under a microscope (40×), and it was found to be fusiform with regular edges, adherent cells, and epithelial-like morphology.
[0070] 2. MDCK-2B2 has a higher growth density
[0071] Inoculate with the same initial density (2.5×10 5 cells / 5mL) of MDCK-2B2 and MDCK in T-25 cell culture flasks, 7 flasks for each, one of the flasks was extracted at intervals of 24 hours for cell counting, counting continuously for 7 days, MDCK-2B2 cells in 7 days The totals are: 1.8×10 5 cells, 2.9×10 5 cells, 11.6×10 5 cells, 22×10 5 cells, 20×10 5 cells, 20.8×10 5 cells and 10.5×10 5 cells; the total number of cells in MDCK in 7 days were: 2.3×10 5 cells, 4.8×10 5 cells, 19.1×10 5 cells, 21×10 5 cells, 23.5×10 5 cells, 24.87×10 5 cells and 14.5×10 5 cells. Such as image 3 As shown, MDCK-2B2 cells hav...
Embodiment 3
[0076] Embodiment 3, identification of MDCK-2B2 cell chromosome
[0077] 1. Harvest the cells, wash the cell wall twice with 0.85% NaCl solution;
[0078] 2. Digest with 0.25% Trypsin-EDTA for 10-15 minutes. When the cell surface appears wrinkled and changes visible to the naked eye, use a pipette to blow down the cells at 453g (1500rpm / min, eppendorf 5810R) and centrifuge at room temperature for 10 minutes. Remove the supernatant and leave about 0.5 ml;
[0079] 3. Hypotonicity: Add 4-6ml of 0.075M KCl at 37°C, blow with a pipette, and bathe in water at 37°C for 3-5 minutes
[0080] 4. Pre-fixation: add 1.5ml ± (methanol: glacial acetic acid = 3:1) fixative, mix gently in a 37°C water bath for 5 minutes, and centrifuge at 453g (1500rpm / min, eppendorf 5810R) for 10 minutes at room temperature. Remove the supernatant and leave about 0.5ml;
[0081] 5. Fixation: Add 8ml± of fixative, mix gently, bathe in 37℃ water for 10 minutes, and centrifuge at room temperature at 453g (15...
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