1.2 When Vero cells were full grown, they were passaged and inoculated into T75 cell bottle, so that the growth density of the next day was less than 90%;
1.3 Add HSV virus into T75 cell bottle according to a certain amount, and continue to culture in 37℃ 5%CO2 incubator;
1.4 Cytopathic effect was observed continuously from 24 to 72 hours, and the supernatant was collected and stored according to the cytopathic effect.
2 HSV virus concentration
2.1 The stored supernatant was filtered with a 0.22μm filter, and the filtered viral supernatant was centrifuged at 50000g for 2.5h at 4℃.
2.2 After centrifugation, the supernatant was discarded, and the precipitate on the bottom wall of the centrifuge tube was resuspended with an appropriate amount of sterile 1xPBS. The resuspended solution was subjected to sucrose gradient centrifugation at 50000g, 2.5h, and 4℃.
2.3 After centrifugation, the precipitate was resuspended in sterile 1xPBS according to the virus titer.
3 HSV virus titers were measured
3.1 Resuscitate Vero cells;
3.2 When Vero cells were full grown, they were passaged and inoculated into 6-well plates so that the growth density of the next day was less than 90%.
3.3 Take a branch of HSV virus and dilute it 10 times according to the gradient dilution method, generally dilute to 10-9. During the dilution process, the virus solution must be thoroughly mixed and evenly mixed.
3.4 The diluted virus solution was slowly added into the corresponding Wells (two parallel samples), 100ul in each well, and incubated at 37℃ for 1h in a 5%CO2 incubator.
3.5 The culture medium in the 6-well plate was removed by suction, then AGAR medium (1.2% AGAR + maintenance medium) was added, and the culture was continued in a 37℃ 5%CO2 incubator for 48 hours.
3.6 Add neutral red medium (0.33% neutral red + AGAR medium), and continue to culture for 24 hours at 37℃ in 5%CO2 incubator;
3.7 The number of leukoplakia was counted under the naked eye;
3.8 The final virus titer was calculated as titer = number of fluorescence x dilution x10.
