Difficult question. you will have to do this by trial-and-error, because the kinetics will depend upon the growth stage of your bacterial culture and state of differentiation of J774 cells. 

can you measure amikacin? HPLC? culture medium supernatant vs very carefully whased cell (lysate) at different times will give you the answer.

Hi Sunil,

The bacteria which is outside the macrophages will easily be washed with the PBS containing your compound. There is not a specific time limit on how long you have to incubate the Macs and bacterial culture to remove the bacteria. Briefly if you are the doing the infection in tube with suspended macrophages just add amikacin in PBS and soon the culture at 1000 rpm for 8 minutes. Since bacteria are lighter than macrophages they will not settle down at 1000 rpm, then you can remove the supernantant and add drug containing PBS and repeat the centrifugation step,  this should remove all the outside bacteria. If you are doing the experiment with adhered  cells then just rinse the surface with the drug by just swirling the plate 3 to 4 times. This should remove all the outside bacteria. I hope this will solve your problem.

thanks to all for giving their time and suggestions

There are 4 variables you need to optimize - 1. concentration of amikacin to kill bacteria using amikacin (is there a general mic value you are looking for?), 2. time of incubation to kill the bacteria at the mic level you expect while minimizing your accumulation inside the macrophage, 3). avoid cytotoxicity of amikacin to the macrophage (should not be a problem if you dont use an extraordinary amount of the drug), and 4) avoid interactions of amikacin with your other trial compounds. I would first figure out what a) concentrations and b) time of incubation would be enough to kill bacteria (at your mic level) in similar conditions as your cell culture. I would then do another experiment with macrophages without bacteria and incubate amikacin at your desired concentrations and time for mic and evaluate post-lysis of the macrophages how much amikacin you can detect through a suitable method, preferentially LC and also measure cell viability using typical cytotoxicity assays. You may find out that you will have to optimize your concentrations accordingly to match these requirements. You will have to also repeat this in the presence of your other trial compounds and confirm that neither amikacin's penetration in macrophage is kinetically modulated by those compounds nor is its cytotoxicity. The first batch of control experiments I mention may already have been published elsewhere, particularly papers in the early 90s. Perhaps you can reduce your experimental load by citing those relevant papers and then performing those procedures. But interactions with your trial compounds may be necessary to confirm.

Aminoglycosides usually do not enter macrophages or other cells even after hours of incubation you should try other antibiotics as chloramphenicol etc. For protocol, you can look at links.

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