Hello!
I am relatively new to what I'm currently working with and as the title suggests, I am currently having some issues with my experiment (viral titration).
So, what I have been doing is the following:
1) Seeding cells on a black grenedier 96-well-plate, by extracting cells from culture flasks with Trypsin, counting and diluting them to a sufficient concentration for seeding.
2) The day after, a virus dilution series was performed in infection medium, and the cells in the grenedier well plate were infected in different concentrations, including negative controls.
3) After incubation, all the cells were fixed using the reagents: PFA, Methanol, PBS.
4) After fixing, the plates were stained using a primary, and a secondary antibody attached to a fleurophore. The primary binds to a viral protein of interest. The antibodies were diluted in PBS, and the second one also with Hoechst. The staining was performed with standard conditions, including incubation and washing 3 times with PBS after each antibody. The plate was stored in a fridge over night before analyzing it.
5) The infection was visualized using a Cytation 5 plate reader.
Now, the issue Im facing is that I have kept seeing some infection in my negative controls, (which should have un-infected cells only), according to the images which showed the flourescent dye in these wells. I am trying to figure out what I am doing wrong.
I have been pipetting from lowest to highest concentration, when adding media or PBS, and used separate liquid plastic containers for the control-wells and the infected ones, to avoid contamination. I am going to redo it once again using new infection media, in case the old flask had contamination.
But apart from this, what other factors could possibly help explain why I keep seeing flourescence from the secondary antibody in my negative controls? ANY type of mistake I could possibly have been making I would love to have pointed out. Or other factors which could cause contamination in the lab.
Could PBS/the other reagents possibly be contaminated as well?
Could it be I am not washing away secondary antibodies sufficiently?
Any tips would be much appreciated!!
There are several possibilities:
- Contaminated reagents/solutions - As you mentioned, if the infection media or PBS was contaminated previously, this could lead to fluorescence even in uninfected cells. Always best to use fresh solutions when possible.
- Antibody binding non-specifically - The secondary antibody may be binding non-specifically to cells, even without the primary antibody present. Try using a higher concentration of serum (e.g. 5-10%) in your antibody diluents to block more effectively.
- Incomplete washing - Residual antibodies left over after washing could lead to background fluorescence. Be very thorough when washing, using plenty of wash buffer each time.
- Cross-contamination - As you noted, be very careful pipetting from low to high dilution to avoid carryover. Also change pipette tips between samples.
- Autofluorescence - Some cells have natural autofluorescence. Make sure you have proper negative staining controls with no primary antibody to distinguish real signal.
- Plate reader settings - Background fluorescence could be exaggerated by certain gain/exposure settings on the plate reader. Optimize the settings using your negative controls.
- Cell density - Higher cell densities can increase background. Try plating fewer cells if possible.
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