You can try this

Thank you Donita for your timely reply. Also, could you please help with regard to how much TCID50 virus should be used for a particular experiment and what does it depend on? I know that the TCID50 is a volume dependent phenomenon. However, someone told me, for a 5 day experiment, 80-100 TCID50 of  HIV-1 strain would be enough. But I do not know whether this is true or how is this calculated. This is because at the end, I cannot select any arbitrary number for setting up my experiment. It should have some explanation as to why 'X' TCID50 would be enough!

Please help! 

TCID50 stands for median tissue culture infective dose, i.e. the quantity of a cytopathogenic agent (virus) which will produce a cytopathic effect in 50 % of the cultures inoculated.

I do not fully understand your need: TCID50 of your stock or how much stock to inoculate in a culture for kinetic study

The paper could help you to get a more specific idea on what others do.

And the paper.

Have also a look on similar questions on RG, you can find valuable suggestions and comments. 

Yes. Thank you Doinita for your valuable inputs and help. I'm currently going through literature, the tcid50 calculation sheet you provided and looking for similar questions on research gate. What I needed to know is that how much TCID50 should I go for when I set up my experiment? And what factors decide how much TCID50 should one use for the experiment.

Basically, we want to look for effect of inhibitor on PBMCs (with the inhibitor concentration showing maximum protection). the PBMCs are pre-treated with inhibitor and infected with virus. We want to carry out the assay over a period of 6-7 days. I do not understand on what basis should I decide how much TCID50 of virus should I use? Or should I go for, may be, trying different TCID50s to see which will be optimum (like 50, 100, 150). We expect to see a protection against infection. But at the same time, do want the cells to survive over a period of 6-7 days. Our seeding density is 0.25-0.3 million PBMCs per well in a 96 well plate (normally this is 0.1 million cells in case of CD4 +ve cells, but in case of PBMCs, we keep it at a higher density).

Our total target volume (volume of virus+ media containing inhibitor and IL-2, PSA, FBS+ PBMCs is 300 microL). So what TCID50 should I go for and why?

To get the chaper you ask for, try to ask the authors:on RG.

David R Miller

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David R Miller 3.43

Mayo Foundation for Medical Education and Research

David R. Miller

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David R. Miller 29.48

University of Connecticut

David O'Reilly

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David O'Reilly 18.80

National Health Service

David O'Reilly

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David O'Reilly 3.61

Monash University (Australia)

Thank you so much, Pasquale sir!

you can use my experiment below,......good luck

The cultured virus was subjected to microtiteration by using flat bottom 96 wells micro plates. The RD cells were then treated with trypsin-versene suspension. After complete monolayer cell culture (24-48), growth media was decanted and cell cultures were washed two times with maintenance medium. Four wells were inoculated with each virus dilution(0.05 ml per each well), and control wells were prepared  for each dilution. After that, plates were covered with a sterile adhesive cover and incubated at 37 °C for 1 hr for virus adsorption. And then the inoculums were decanted and 0.1 ml of maintenance medium was added  per each well. Plates were covered again and incubated at 37 °C for 48 hr followed by examined under inverted microscope for the appearance of CPE.

Viruse titer was calculated according to the protocol of CVB/NVSL,(2000) as follows:

Titer =(X-d/2+(d×s)) were:

X: log 10 of lowest dilution &=0

D: log 10 of dilution factor&=1

S: sum of proportion of CPE positive divided by number of wells inoculated for each dilution.

For testing an inhibitor you want to make sure the cells in each well will be infected. Using TCID50s you cannot just double the volume and assume virus will get into each cell. You need to be in excess to assure each well will have virus and enough virus to infect PBMCs present. TCID50 depends on the number of cells in a standardized tissue culture assay. For you it sounds like this is based on 10^5 CD4(+) cells and you are now using 3x10^5 PBMCs per well. That's 3 x more. Without empirically determining the TCID50 on PBMCs you should also allow a little extra virus to ensure all PBMCs will infected. So I would think 50-100 TCID50 should be a good place to start. This assumes that you want the target of "all cells infected at the beginning of the experiment" in order test the potency of the inhibitor. 

Thank you very much for your answer A J Roberts ma'am! I'll do as you said and see if it works.