Can anybody suggest to me that what would be the best way to check bacterial phagocytosis by macrophages via imaging?
1. One of the best way is to take fluorochrome tagged bacteria and proceed for confocal microscopy. Else fluorescein-conjugated bacteria can be used.
2. Use GFP tagged bacteria (Transform).
Hey Shivani,
As Sanpreet already mentioned, analysis with bacteria that fluorescent by themselves is the best method as well as using a confocal microscop. A few more suggestions:
If the fluorescent bacteria are not an option you can stain bacteria with commercially available antibodies, but of course not for every bacterium there is an antibody. For the sole purpose of phagocytic behaviour of macrophages I would use an avirulent E.coli strain (e.g. K12 DH5-α) and make a small kintetic (0, 15, 30, 60 min). For a detailed protocol, including infection, fixation, staining, confocal microscopy and data analysis, you can have a look at our paper, if you like:
Article The β2 Integrin Mac-1 Induces Protective LC3-Associated Phag...
Most of it was performed with Listeria, but there are also experiments with E.coli and B.subtilis.
Please feel free to ask any further questions,
all the best and stay healthy,
Marc
It depends on your scientific question. If you more interested in macrophage behavior fluorescent zymosan particles such as Phrodo-Green or Phrodo red are a great substitute. Zymosan is a fragment of the bacteria wall (E. Coli) so the macrophage response is identical to live bacteria, an added benefit is that particles become fluorescent only when absorbed by the cells. You also don't need to deal with live bacteria in the mammalian cell culture in the incubator (i.e. cross-contamination risks, risks of bacteria overgrowing the macrophage culture). On the other hand if you interested in the bacteria itself and not the macrophages (i.e. bacteria survival or resistance to macrophages) I suggest you simply incubate the bacterial pellet with Rhodamine or FITC that will make the bacterial transitory fluorescent and seriously consider using a separate incubator for this work that you can decontaminate properly after completion of the experiments. Secondary fluorescent antibodies against your type of bacteria will work as well but keep in mind that many antibodies have antibacterial components in them such as Sodium Azide and Trehalose that are particularly toxic to bacteria. Again if you are studying bacterial behavior I advise against transforming them as any data you obtain may be criticized as nonreliable due to the effect of transformation.
Hi, thank you Sanpreet Singh Marc Herb and Anton Lennikov. These are really nice suggestions but I have resource limitations so I cant use antibodies or flourescent tags, they would be costly affair.
I do want to use fluorescent dyes to use in my experiments but I have faced the problem of leakage and non specific, background signals. I have tried labelling bacteria with AO, DAPI, PI but results were not satisfactory. I am working with S. flexneri and getting a flourescent tagged bacterial plasmid for S. flexneri would really be a great help for my experiments. I am intrusted in behaviour of macrophages more upon drug treatment so trying dextran or zymosan could be effective but It will make more sense for my work if I do my work with S. flexneri.
May anybody suggest me please that if DAPI or AO or PI could be used for labelling bacteria and how should the fixation be done? Would bacteria lose dye after fixation?
You can use Bactoview https://biotium.com/product/bactoview-live/ to perform fluorescent tagging of the live bacteria there are red and green versions for 488 and 595 nm channels. If you really on a tight budget ethidium bromide can do the same thing. https://jcm.asm.org/content/jcm/19/4/453.full.pdf here is the manuscript from 1983 describing the use of EthBr to detect the bacteria in the blood. You can observe the Ethidium bromide fluorescence at the DAPI channel if you can you should adjust your emission filter to accommodate the EthBr emission peak at 610 nm if your fluorescent microscope allows it. If not the DAPI filter set will miss the peak but you still should get decent signals. In terms of the effect of fixation, you can probably use methanol, but you need to check how it will affect the fluorescent signals of your dyes.
Carboxyfluorescein succinimidyl ester (CFSE) is a fluorescent cell staining dye. CFSE is often used to track cells (e.g. during phagocytosis) or monitor cellular division. It appears in the FITC channel and is easily detectible by fluorescence microscopy or flow cytometry. CFSE is able to passively diffuse into cells, making CFSE staining a good method to visualize phagocytosis of bacteria. CFSE-stained bacterial cells are an extremely affordable and effective macrophage target.
Moreover, for quantifying phagocytosis of any kind of bacteria, you can stain it by FITC.
Thank you Anton Lennikov and Vinod Nadella for suggestions. I will try with Etbr and CFSE looks like a great choice. Would check for the price and see if I can get it. Thanks a lot for these great suggestions.
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