I need to isolate a tiny fraction of macrophages that could successfully kill the internalized bacteria (for example, M. tuberculosis). I think the best way is FACS, but I need fluorescent dye(s) that deferentially stain these two populations, while keeping their RNA content intact so that I can perform RNA-Seq.
This actually might be tougher than you think.
1. First you have to sort out the infected macs versus the non-infected ones first. This is the easy part since you can sort based on the fluorescence of the internalized MTB. Because not all macs will be infected...
2. And this leads to the second issue...which is MOI. The higher the MOI, the lower the chance of mac survival. So...the ability to kill internally is also related to MOI.
3. If we don't look at mechanism, and just visually. The only way that I know you can differentiate between macs that can kill versus those that can't is to find the MTB co-localized to the lysosome. This is usually by microscopy... I'm not sure you can actually do this on the sorter...if you have a sorter that can also take pictures of the single cells (which I don't know if they exist)...maybe
4. The only other option is a dye that changes between live and dead. But I don't think these exist... The dyes either label the live cells or the dead cells... but what you would need is a dye that when in live MTB is one color and when the MTB dies, it changes...
5. Another option is to find a surface marker that correlates with macrophage killing activity (I'm pretty sure it has to be surface because internal staining will make the cells not viable)... And sort among just the infected cells for this marker... The only thing I can think of for a marker is a MHCII tetramer for a MTB peptide (such as Ag85B). But even then, it is more than likely you will miss macs that doesn't present that peptide...
6. The last point... I'm not sure the intracellular killing is an all or nothing event, meaning that I don't think you can say that one population of macrophages can kill while another can't, not if they are all treated with one condition. I think it is more than likely you will get populations that can kill, that can't kill, and everything in between.
Hello,
can you have access to fluorescent bacteria? In combination with a marker for activated macrophages you could sort 3 populations:
- non activated macrophages non fluorescent
- fluorescent activated macrophages with fluorescent non degraded bacteria
- fluorescent activated macrophages without fluorescent bacteria (degraded)
Hi,
like befaure mentioned: dsRed or msCherry bacteria are working very well...gfp not so good.
and then sorting.. :-)
Attached: msCherry bacteria witin macrophages (or GFP; MOI 10)
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