TCID50

[ryanmichaeljohnson]

I am new to this field so please be simple in answering this question.

I know the TCID50 of a HIV virus stock. I now wish to use this stock to test drug resistance. If my TCID50 is 1x10^7 TCID50/ml, how much volume would I add for well of 10 ml of MT2 cells previously exposed to a drug? The cells have been washed of the drug and the concentration of the cells is 2x10^5 cells/ml.

I calculated the virus stock using a paper by Johnson and Byington and they state that my TCID calculation may vary by a log.

I am a little lost because I am not seeing how to convert from TCID to moi and then what is the best amount to use to calculate my concentration.

Thanks,

Ryan

[protold]

Hola Ryan, Difficulty of your question makes,that you haven´t any answer yet. I work in baculovirus and need a manual to help you . TCID50 is the dilution giving a response greater than the 50% positive,so you will have non infected wells, (to test a drug you need 100% of infection, I think ).For that you need to know the virus titer and use an elevated multiplicity of infection,wich is the number of virus that you inoculate by each cell. Look in your institution for the manual of baculovirus of O´Reilly where the mathematic development of end-point dilution is made.
I reproduce the most important steps:
log TCID50=log dilut.giving a response greater than 50% - proportional distance of that dose => negative value.
The virus titer is the reciprocal of TCID50/ml. Multiplicating by a factor coming of a Poisson distribution of 0.69, you obtain the pfu/ml ( plaque forming units/ml) wich is the real value of viruses in your stock.
If your value 10E+7 is TCID50/ml, the title will be 6.9x10E6, If you have a suspension of cells 2x10E5/ml in a multiple plate of 24 wells to 0.5ml each you have 10E5 cells/well; inoculating with 15 ul. you have an MOI=1 aprox.
I hope have been of help. Please look the manual and good luck

[protold]

Sorry, for 10ml 2x10E6 total cells and a title of 6.9x10E6 pfu/ml, with 1 ml of inocule you have a MOI 3.4. For a MOI=1 you have add 290ul. Bye

[ryanmichaeljohnson]

Potold,

thanks it does help.

Ryan

[Myotis_rufopictus]

When I was in grad school I made a "cheat sheet" and taped it to the wall in our cell culture/virus room...for those days when we hadn't drunk enough (or sometimes, too much) coffee.

It said something like:

Titer in PFU = 0.69 * titer in TCID50

MOI = viruses / cells

To reach desired MOI:
Volume of virus to use = Cells * (MOI / titer in PFU)

By the way, here's a link to a TCID50 calculator as a Microsoft Excel spreadsheet. This is what my grad school lab used to use.

[ryanmichaeljohnson]

Thanks!!