The virus titer was calculated based on the number of plaques.
Experimental reagent
1. 4% agarose gel: 2g agrose 50ml water, autoclaved
2. 2×Grace(unsupplemented): 9.14g powder (invitrogen) 0.07gNaHCO3 H2O, adjusted to pH6.1, constant volume to 100ml, sterile filtration
3. supplemented Grace: 1 × Grace added 3.33mg/ml Lactalbumin, 3.33mg/ml yeastolate, sterile filtration
4. High temperature inactivation of FBS
experiment apparatus
1. 6-well plate
2. 12ml centrifuge tube
3. 100ml sterile glass bottle
4. Sterile straws
5. 70 ° C water bath
Experimental Materials
Baculovirus, SF9: 5 × 105 cells / ml
Experimental procedure
1. Under sterile conditions, 2 ml/well cells (5×105 cells/ml) were seeded into 6-well plates, incubated for 1 h at room temperature to adhere to the wall, and the degree of adherence was examined by microscopy after incubation;
2. Dissolve 4% agarose gel in a 70 ° C water bath, empty 100 ml sterile bottle and 2 × Grace into a 40 ° C water bath to preheat;
3. Dilute the baculovirus with a serum-free supplemented Grace gradient: 10-1~10-8.
4. Discard the supernatant in a 6-well plate, quickly add the diluted virus, 1 ml/well, and re-well, incubate for 1 h at room temperature.
5. Configure the upper agar: add 20ml high temperature inactivated FBS to 2×Grace 100ml, 25ml 2×Grace (including FBS) 12.5ml sterile water 12.5ml 4% agarose gel, to preheated 100ml sterile bottle, gently mix Evenly, put in a 37 ° C water bath for later use;
6. Discard the supernatant in the 6-well plate and quickly add 2ml of the upper agar to prevent the bacteria layer from drying and let it stand for 10-20min to solidify it;
7. Place the 6-well plate in a 27 ° C incubator for 4-10 days;
8. Count the plaques in the plate until there is no change for two consecutive days, add 0.5 mg of neutral red 0.5 mg, and incubate for 2 h at room temperature to count plaques.
Note:
Virus titer (pfu/ml) = 1 / dilution factor × plaque number × 1 per well inoculation volume
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