Hello, I'm a graduate student working in a lab as part of my degree.
I am currently trying to calculate TCID 50 values using HIV pseudovirus and TZM-bl cells. To my understanding, I need to score wells as positive or negative using a luciferase activity cutoff, however I don't understand how a cutoff is chosen. I found a few articles using a cutoff 2.5 times or 3 times the signal of the cell control wells. Can I simply use a similar multiple, or is there a way to calculate the best cutoff each time?
I have attached the articles in question with relevant sections highlighted.
I would use the signal of the control wells plus/minus 2 standard deviations. In clinical settings 2sds is generally used for all assays for an acceptable control range. I would calculate this range based on several days worth of controls to account for day to day variation.
As the luciferase activity is very variable per se, you may get a big SD. So it is recommended to repeat the assay more than three times to get a cluster for the luciferase activity. Having more number of experiments help to identify the outliers in your assay. Then you can use MEAN ± 2SD tp set your threshold.
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