macrophage infection assay - problems with macrophge infection assay (Jul/13/2010 )

Dear all,

I am currently testing some new antibiotics which are thought to be active against TB. After the in vitro assay and the MIC determination, I now wanted to check out the action of the antibiotic on a culture o Macrophages infected with TB. I have encounterd a bit of a problem in trying.

The protocol I use is briefly;

- Pregrow J774 cell line
- Wash the cells and count them with a coultercounter
- Place +/- 250 000 cells in 1 ml DMEM without gentamicin in each well of a 24 well-plate
- Leave the cells for incubation for 1 hour at 37°C 5%CO2 to adhere
- Put TB desolved in DMEM without gentamicin onto the macrophage culture wit an MOI of 10
- Leave the cells for incubation for 1 hour at 37°C 5%CO2 to infect
- Wash the cells with DMEM without gentamicin
- Add the antibiotics to the wells (each condition is done in triplicate)
- At the right timepoint, I remove the meduim rom the celculture
- Lyse the macrophages with 200 µL PBS 1% triton or 15 minutes
- Add 800 µL o PBS to each well and mix
- Take from each well 200 µL of the PBS/lysate and add it to 800 µL PBS in an eppendorf (I do this twice and take the average of both measurments)
- Add 100 µL ethanol 1% decanal and Measure the RLU.


The problems I have is that even in the control samples, when the measurments at day 0 and day 1 are compared, there is a large reduction in the RLU. The mycobacterium seems to have disapeared with almost a log10 difference.
The second problem is the triplicates tend to differ quit a bit and even *remeber I measure each sample twice* both measurments of the same sample differ.
The third problem is that I I measure the RLU of the supernatants which, I remove frome the macrophagecultures before lysing them, I have a high count of TB in that supernatants, so extracellular TB. The thing that is realy annoying about the latter problem is that the testresults are not so consistant and in the supernatants the antibiotics tested kill off fewer TB than in the in vitro assay.
I know that the first problem could be taht my J774 macrophages are activated by LPS pressent in the medium, using Bone marrow derived could solve this, however this would become to cumbersome to test compounds on a large scale. The high count of micobacteria in the supernatants could be due the cytotoxicity o the compounds I use, but it remains strange that in the in vitro assay the antibiotic just simply kills the mycobacteria at the same concentration.

Does anyone have some experience with this kind of infection assay, encounterd the same problems? Anyone has a better protocol and is willing to share?
If someone could give some advice, that would be very helpull!

thanks in advance!

kind regards

Davie

Ok, So I already changed some things in the protocol.

-I will use a lower MOI of 3 or 1 because a MOI o 10 apprently reduces the macrophages to crap
-Instead of using glycerol stock I will use a live growing culture of about OD 0.6
-Wash a few times more with DMEM before incubation with antibiotics

please feel free to give suggestions.

best regards


Davie