I'm doing experiments with M.tb/M.bovis infected macrophages and allow 4hrs for the macrophages to phagocytose the bacteria. What is the minimum time I can allow for phagocytosis to happen?
It happens pretty quickly- I used to do 2h infections and they seemed to work fine, and I didn't get any differences in #'s when compared with 4h or overnight infections- although these were unactivated J774 cells with Mtb (H37Rv)- so maybe different with RAW246.7- even though still a murine line, and if yours are activated, and with Mb instead of Mtb. If you are really interested you could check with microscopy?
You may reduce infection time while maintaining infection rate by using the 'spin-infection protocol: perform infection in a plate ( 6-well, 24-well ), add bacilli to cells and spin plate at 1000 rpm for 10 min.
That is an interesting question. In fact, you can infect mouse or human macrophages (either cell lines or primary cells), with M. bovis, M. tb, M. avium, M. smegmatis, and other mycobacteria, from as early as 5 minutes to 4 hours. If you infect during 5 minutes, you will have a lower rate of infection. However, if you infect longer, you will reach a plateau of maximal infection rate. Using 1 h of infection, I achieved that plateau with M. smegmatis. For M. tb, it took me 4 h, in J774 cells. Therefore, the bacterial species is an important factor for determining the minimal infection time.
The infection time also depends on the macrophage cell line. The best way to know the minimum time for infection, is infecting your cells with your strain of bacteria, and then, quantify it. If you have a bacteria with a GFP tag, you can quantify the infection rate by flow cytometry, fluorescence microscopy or confocal microscopy (see the pdf attached).
The most important thing, is to have a perfect bacterial suspension without clumps, from a mid-log culture. You should have a single-bacterial suspension. Otherwise, you will stress the macrophages and make a non-reproducible experiment.
Finally, the multiplicity of infection (MOI), if is very important in defining the minimal time for infection. You should also test several different MOIs, to optimize your infection protocol. Higher MOIs of M. tb, increase the infection rate, but kill the macrophages as well. So you should reach a compromise between the highest possible MOI and the longest infected-macrophage survival.
I would like to comment the last message. If you spin at a lower speed (1000 rpm), like suggested by Martin Gengenbacher, you will bring down only the large clumps, and most of the single bacteria will remain in the supernatant. You can test that easily, by spinning 10 ml of a bacterial suspension at 1000 rpm, for 10 minutes, and then measure the OD (600nm) of the supernatant. You will see the cloudy supernatant indicating a bacterial suspension. To lower down all the bacteria, you will have to spin at 3500 rpm. Using this higher speed, you will see a clear supernatant, without bacteria. You cannot apply 3500 rpm to the macrophages because they will all die. So, spinning its not an effective method to infect macrophages with a single bacterial suspension of mycobacteria.
If you infect by using the smallest possible volume of bacteria, you will not need to spin down.
Chapter Application of Confocal Microscopy for Quantification of Int...
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