I need to carry out phagocytosis assay in RAW 264.7 cells using E.coli GFP for microscopic visualization.
1. How do I calculate bacterial number for the assay that can help me determine the MOI. I understand the basic method is serial dilution. Is that what is followed each time when people carry out assays? or is there some other easy way to do this?
2. If someone can share the protocol they follow for such experiments, it would be immensely helpful.
I am completely new to doing such assays and any help or suggestion is highly appreciated. Thank you.
The normal and the usual method is to titrate the bacteria (0.5-50) and use the MOI that best shows the differences between the treated groups in comparison to control.
If your idea is to just see E.coli GFP uptake by RAW 264.7 cells for microscopic visualization, try MOI 10 and that should give you a good view !
Uptake is also defined by cultural conditions and duration of incubation.
Following the duration of incubation, wash the cells with RPMI and incubated in experimental medium containing gentamicin (100 μg/ml) for 1 h to kill the extracellular bacteria.
Thank you for your suggestion Vinod Nadella
Do you have a detailed protocol to share? I plan to check for microscopic visualization as well as try and quantify the bacterial uptake and eventual degradation.
Also, how do you count the number of bacteria. I am fairly new with this so don't know how to start out. Any suggestion is welcome. Also, when E.coli is incubated with RAW, does the media have to be without antibiotics?
Thank you again!!
Seed RAW 264.7 cells at 5 x 10^4/well in a 24 well plate. Following 24h incubation, infect the macrophages with E.coli at MOI of your choice in medium w/o antibiotics for a defined time period. Following incubation, remove the culture media and wash the cells extensively with fresh media to remove extracellular bacteria. For better results culture the cells in gentamycin (10 μg/ml) containing medium to remove extracellular bacteria.
Taking advantage of the GFP tagged E.Coli, bacterial uptake can be analyzed by flow cytometry by getting the macrophages in suspension. This can be achieved by treating the cells with 0.05% trypsin and/or 0.5. mM EDTA. Cells can be fixed with 4% paraformaldehyde
For visualizing the bacterial uptake, you can use fluorescent imaging. Cells can be fixed with 4% paraformaldehyde. Cell Nuclei can be counterstained with DAPI. This will give you the opportunity to quantify number of macrophages positive for E.Coli and number of E.coli taken by each macrophage within the zone of visualization. Phagocytic index can be generated by multiplying the percentage of phagocytosis by the mean number of internalized bacteria per cell.
For tracking bacterial update, try live imaging.
Also, I recommend below article for for visualizing degradation of E.coli within Macs.
https://www.cell.com/cell-host-microbe/pdfExtended/S1931-3128(17)30195-6
When I worked on these phagocytosis assays, I used non-fluorecent E.coli. So, I lysed the cells after a defined time period for phagocytosis. Cell lysates were serially diluted and plated on Luria agar plates. 24h post incubation, colony forming units (CFU) were counted. This gives the number of e.coli taken by macrophages under different treatment conditions. This method was also used to study e.coli degradation within Macrophages by collecting the cells lysate after a defined time post phagocytosis. Difference in the CFU count will define the e.coli degradation within macrophages under different treatment conditions.
BW
Hello Ruchi, try https://academic.oup.com/clinchem/article/54/5/911/5628451they use gfp-bacteria to access phagocytosis. BD supply a PHAGOTEST - fitc opsonised E.coli with instructions https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.gb.341060.pdf
Hope this resolves any issues.
Vinod Nadella
Thank you so much for the detailed protocol. It is extremely helpful.
To determine the number of bacteria to be used for MOI, do you follow the OD600 = 1 for approx 10^9 cells or CFU/ml by carrying out serial dilutions?
I apologize for this barrage of questions.
Thank you again!!
Depending on what MOI means, either way make sense !
If you are after MOI 10, the 10CFU per 1 macrophage. All it depends is how you come to this final required ratio.
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