I'm working with Herpesvirus and Adenovirus. For Herpesvirus, I use Vero cell line to activate and check virus titer. For Adenovirus, I use Hela cell line.
Recently, I could activate them, but can not have high titer. The titer i need around 10^9 or 10^10 TCID50. But I just have around 10^5 TCID50.
I use this protocol to activate virus:
1. Cell cultured in 75cm2 flask for 2- 4 days, until 90 - 10% confluent.
2. Wash 2x with PBS.
3. Infect 3ml of virus stock for each 75cm2 flask for 90mins, rock the flask every 15 mins to deposit all cells in flask.
4. After 90 mins incubation with 3 ml of virus stock. Add more 20 ml of medium (DMEM+2%FBS+1%P/S + 44mM Sodium bicarbonate).
5. Incubate flask for 2 - 3days until 90 - 100% CPE appears.
6. Freeze (-70 degree) and thaw (4 degree) 3x to lyse cell.
7. Cfg 1500xg, 4 degree, 10 ml, collect supernatant for virus stock.
Could you please show me specific protocol to increase these viral titer? Thank you so much.
What is in your virus stock? I would suggest checking the virus concentration and calculating how many ul virus you need for different MOIs, and then maybe infect flasks with increasing MOI to see how much is needed. Dilute X ul of virus stock in minimal amount of serum-free medium to cover the surface (this works in our lab). Incubate for 1h in 37C incubator and rock every 15 minutes. Then wash with, and add your normal cell medium; we use DMEM with 10% FBS and 1% Pen-Step. Incubate for 36 to 48 hours and harvest according to purpose.
Thank you so much for your quick reply. Amanda Westergren Jakobsson. I really appreciate your help. In my case, I infect 3 ml of virus stock (titer is 10^5 TCID50/ml) for 90 mins and rock every 15 mins. After that I did not remove that amount of virus and did not wash. I add directly 20ml of medium (DMEM+2%FBS+1%P/S + 44mM Sodium bicarbonate).
For MOI, i used 75 cm2 flask, the concentration of cells for infection is approximately 10^7 - 5x10^7 cells/flask. 3 x 10^5 TCID50 / around 3x10^7 cell = 0.01. Is this MOI enough for infection?
After infection for 90 mins, i did not wash. Does it affect to viral infection?
Tran Xuan Ngoc Huy, for the MOI you can go up to 5, 50 and even 100 - 0.01 is very low! I'm not sure what the effect would be of leaving the virus in the plates, but working with high titers it sounds very stressful for the cells. After infection we want to relief the cells of any stress and provide them with the nutrition and energy that they need for the virus to replicate and do its thing. The Adenovirus hi-jacks the cellular network and is dependent on cellular functions.
Amanda Westergren Jakobsson . Thank you so much. I will follow your instruction and optimize again my own protocol.
Usually, how about the concentration of cell in flask do you use for infection? I mean the confluent of cells? 70%, 80%, 90% or 100%. Since base on your knowledge " After infection we want to relief the cells of any stress and provide them with the nutrition and energy that they need for the virus to replicate and do its thing.". I think the cells in flask should be at moderate concentration (70- 80%, but must be ensure the compatible MOI), with this, they can proliferate more, and simultaneously virus also can propagate inside the cells. And the titer can be increased. Am i right?
Sorry for asking many questions. Thanks.
Have a nice day and be safe with covid 19.
Tran Xuan Ngoc Huy Yes, we go with a confluence of 80-90% before infection. Good luck!
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