My negative control (only cells non stimulated stained with FLICA) is stained as my positive control (LPS+MSU cystals).
Does anyone have any idea what should I do in order to avoid it?
The protocol says:
8. Incubate 30-60 minutes at 37°C, mixing gently every 10 minutes.
9. Wash by adding 2 mL 1X Apoptosis Wash Buffer.
10. Centrifuge at 200 x g for 5 minutes.
11. Aspirate supernatant and resuspend cells in 2 mL 1X Apoptosis Wash Buffer.
12. Incubate 10 minutes at 37°C to allow any unbound FLICA to diffuse out of the cells.
I did all they asked to but didnt have any success :(
Thanks in advance :)
Quantification of immuno-fluorescence (IF) is a challenge in the assay. Therefore, without knowing the approach that you are using to compare the IF signal between "control" and "treated" cells, it is harder to diagnose the problem. The best approach would be to perform immunoblotting for activated caspase-1 that is present in the cell lysates and secreted in the medium.
Jordan, I used peritoneal macrophages (adherent cells) and I performed a flow cytometry analysis.
Hi Nathalia, did you resolve your problem with Flica? I started with assays and I have problems too with positive signal in control.
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