I am running a focal reduction assay and having some background issues when I read my plates on my CTL plate reader. I will describe the assay briefly to see if somebody has some incite on how to reduce the cell control background (I don't always see the high background and haven't really been changing my assay conditions):
1. Infect confluent MDCK cells with influenza virus (2Hr) in 96-well plates (media contains 0.1%BSA,TPCK-trypsin)
2. Add Avicel overlay and incubate plates overnight @37C.
3. Remove overlay and wash plates 1X with PBS.
4. Fix for 30min with cold 4% formalin (in PBS).
5. Remove fixative and wash 2X with PBS.
6. Permeabilize cells with PBS/Glycine +0.5% Triton X-100 (20min).
6. 3X PBS/Tween (0.05% Tween-20).
7. Incubate 1° Ab diluted in ELISA buffer (PBS,10% Horse sera, 0.1% Tween-80) for 1Hr
8. 3X PBS/Tw wash
9. Incubate 2° Ab in ELISA buffer for 1hr
10. 3X PBS/Tw wash
11. Add substrate (KPL True Blue) and Inc. 10min @ room temp
12. wash with tap water and dry plates
13. read on CTL reader.
Thanks
I think you should use other blocking buffer (BSA/FBS/TWIN-20) for permeabilization of cells and further antibody dilutions (first and second antibody). Regrading after washing substrate addition does not make any difference, just you need to blot your plates properly on the Whatman blotting paper. Also you can get read of background in the CTL biospot analyser using different gating system.
I did at least 15 plates per experiment/ day and haven't had any problem.
Good luck
I have figured out the issue of the background. It appears that the cell concentration in the well was too high and when I reduced the cell concentration this has drastically reduced the background signal in the wells.
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