1. Preparation of cells
a) Resuscitation 293 cells, subculture for 1-2 generations, adjust the cell state, and complete the transfection experiment within 4 generations.
b) 1 day before transfection, trypsinization was passaged, 5×105 cells were seeded in 6-well plates or 87.5px dishes, and 2 ml complete medium ( DMEM+10%FBS+1%P/S+1) was added. %Glutamax ), shaken and placed in 5% CO 2 , 95% humid air, 37 ° C incubator to ensure that the cell density reached 70% on the next day of transfection.
2. Transfection
a) On the day of transfection, the medium was removed from 293 cells and replaced with fresh 1.8 ml DMEM + 10% FBS medium (without antibiotics) and incubated for 2 h at 37 °C.
b) Preparation of transfection reagent mixture
c) Use transfection reagent POLO3000:
d) tube A: mix 4 ug DNA with 10 0 ul D MEM (basic) ;
e) B tube: Mix 6 ul POLO3 000 with 10 0ul D MEM (basic) ;
f) After standing for 5 min at room temperature, mix the A tube with the B tube solution, gently mix and incubate for 15-20 min at room temperature;
g) The above complex was uniformly added dropwise to 293 cells, and the cell culture plate was gently shaken and incubated in a 37 ° C incubator.
3. Adenovirus packaging
a) The cell state was observed after transfection for 24 to 48 hours. If the plasmid was fluorescent, the transfection efficiency was observed under a fluorescence microscope, and the fluorescence rate was about 10-70%. A cell density of >90% is required for the turntable, and the cells are all digested with trypsin and transferred to a 250px cell culture dish.
b) After that, observe the state of the cells every day, change the medium every 2 days, change the liquid 8ml each time, until the lesions (CPE) appear, then change the liquid to take a half change, then leave 3ml, then make up 5ml.
c) On days 7-10 after passage, the cells showed 60% CPE in a 10 cm dish, most of the cells disintegrated, and the cells and culture were collected.
4. Preparation of crude adenovirus
a) The harvested centrifuge tube containing the cell culture supernatant and cell debris was placed in a -80 ° C refrigerator. After 30 min, the tube was placed in a 37 ° C water bath and thawed for 15 min, so that it was repeatedly thawed twice to lyse the cells.
b) The frozen and thawed virus solution was centrifuged at 3000 rpm for 15 min at 4 ° C to remove cell debris.
c) The crude virus extract 1 ml/tube was dispensed into a 1.5 ml centrifuge tube and stored in a -80 °C refrigerator.
5. 3.5 Adenovirus concentration
a) The virus supernatant of the previous step was filtered through a 0.45 um cellulose acetate membrane, and the wetted membrane was filtered with 5 ml of DMEM complete medium before filtering the virus solution to reduce the adsorption of viral proteins by the filter.
b) The virus solution after filtration was placed in a Beckman high-speed centrifuge tube, centrifuged at 50,000 g at 4 ° C for 2 h.
c) After centrifugation, take the tube back into the safety cabinet, carefully pour the supernatant, and fold the tube down on the absorbent paper to drain the residue.
d) Take 1ml of pre-cooled DMEM, add it to the centrifuge tube in turn, cut off the tip of the tip, repeat the half-blowing and sedimentation repeatedly, gently operate, and completely resuspend the sediment at the bottom of the centrifuge tube in DMEM solution. The tubes were rinsed again with 1 ml of DMEM to obtain 2 ml of virus concentrate.
e) Store a small amount of 100ul per tube and a special tube for virus storage at -80 degrees.
Continuous release...
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