Hey Larisa,

we also did cell viability/death assays with infected macrophages (PM,BMDM + Listeria) and tried different methods.

The most classic one is the LDH release. I know it is lytic cell death detection, but it is easy to perform and gives you a good hint. Some bacteria also secrete LDH, so in that case you have make control wells just with your bacteria and medium inside to check if they produce a background signal. When you have no or low levels in comparison to your macrophage wells or the positive control (Triton or lysis buffer), then you are good to go.

Another option we tried for living cell detection was the CyQuant Kit. It worked very well, but here we had the same poblem as you, because the dye also binds to bacteria (or all that is living and contains DNA :D). So we made controls for it. For every conditon we had on the plate, we made Triton control. That means that you lyse your cells completely with triton and get only the signal of the bacteria inside. This signal can be substracted from your normal wells. Sounds complicated, but it is not and worked very well.

If you like, I can send over the protocol and an example plate scheme for you. For an example how it looks, you can have a look at our publication.

Article Mitochondrial reactive oxygen species enable proinflammatory...

I would refrain from Kits like CellTiter-Glo for viability. Most people use it for cell viability because if a cell dies ATP levels usually drop too.

However, we observed that, while ATP levels dropped over 1, 3 and 5 hours after bacterial infection, the macrophages did not die during that time (cell death was analyzed via other techniques). That means that reduction of ATP levels (which you measure with CellTiter-Glo) does not necessarily mean that also your cells are dying (they can just use up a lot of ATP).

All the best and stay healthy,

Marc

Dear Mark,

Thank you very much for your advice! It is very helpful. I'm thinking about option 2, making Triton control. Can you please send me your protocol how you did it? If I understand correctly, I could use PrestoBlue instead of CyQuant Kit and I treat all of my cells the same, then I add PrestoBlue and measure the signal, and then I could remove Presto, lyse my cells with Triton and add Presto once again and measure the signal. And then I just substract this signal from my signal with cells.

I look forward to hearing from you.

All the best to you too,

Larisa

Hey Larisa,

hmm I think it is not that easy. In the Cyquant Kit is a nucleic stain, which marks all of your cells, and a supressor, which covers all dead cells. So you detect with green fluroescence only living cells. You add these substances together at the end of your incubation time, incubate, wash and then add the triton and measure. I will translate the protocl to english and send it over to you together with a plate scheme :)

All the best and stay healthy,

Marc

Thank you! And you measure the fluorescence? We have Cytation 5 (Biotek).

Best regards,

Larisa

Hey I have send you the protocol and an example of one of our experiments. Any plate reader which can detect fluroescence is fine.