- I seed 24 wells TCP with a seeding density of 5x10^5 cells, and let them grow for 24 h.After 24 h they reach a cell density of approximately 70-80%. I infect them with my bacteria, and after completion assay, I recover significant number of bacteria that invaded the cells.
- However when I let my cell lines grow for 48 h (Confluency >90%). Very few number of bacteria are able to invade.
- I see most of the papers mention confluency but no time period of incubation. So I am not sure at what conflunecy its favourable to infect the cell lines 70-80% (after 24 h) or (90% after 48 h)?
Looking forward for kind replies.
Clearly, you bacteria require some sort of growth of cells and not quiescent cells. So I would definitely drop the confluency to 50%, or stick to the 70%.
Don't estimate confluency by eye. That is quite meaningless. One persons 50 is another persons 70 !!
Count the cells before you plate them. Then after 24 hrs see which cells number gives the 'confluency' you desire. Then every time you plate the same number of cells you are always right.
Good luck!
Some cells differentiate when they become too confluent which can alter bacterial invasion. Confluency of 80%-90% is commonly descripted in literature for bacterial invation. If your seeding density gives you 70% after 24h you could change it to reach 80-90% after 24h and see what happens with invation then.
Hello Zara. How do you recover your invaded bacteria? Let's see if the issue was from the protocol. What was your experimental control to ensure the bacteria should grow?
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