I will be infecting HeLa cells with C. burnetti. The MOI is supposed to be 50. Unsure how to go about calculating how much of the pathogen to add.
I have 1x10^6 Hela cells per well (in 3 mL of media) and the bacterial stock is 1.81x10^6 GE/mL.
I'm not familiar with using "GE" and not sure how to calculate. Thanks!
Hey Casey,
the formula is always the same:
Concentration of your bacterial stock (cells/ml) divided by
Number of cells in well x MOI x Dilutionfactor of your infection volume
I would recommend to remove the 3 ml medium and infect in 1 ml or less depending on well size. We infect with 1 ml for 6- and 12-wells, 0,3 ml for 24-wells and 50 µl for 96-wells.
Lets say you infect with 1 ml per well. So in your case it would look like this:
1,18x106 bacterial cells/ml divided by
1x106 cells x MOI 50 x 1 ml infection volume
This gives you a dilution factor of 0,0236 for your infection solution, which is not very nice to pipette.
You can solve this very easily by increasing your bacterial stock solution concentration.
We dilute our bacterial stocks always to 1x109 cells/ml.
An example from our lab would look like this:
1x109 Listeria/ml divided by
500.000 macrophages x MOI 5 x 1 ml infection volume (in a 12-well)
The dilution factor for your infection solution would be 1:400.
Lets say we want to infect 4 wells, then you need at least 4 ml total. We take 5 ml as spare volume and then you get 5 ml divided by 400 = 12,5 µl bacterial stock in 4987,5 µl infection medium. Then you deliver approximately 5 Bacteria to each macrophage in the well in a volume of 1 ml.
In your case, I would not dilute your C.brunetti culture so much.
I hope this was not explained to complicated. Please ask for further explanation.
All the best,
Marc
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