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 flourescence 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!! :/

  • $\begingroup$ Welcome to the biology stack. Please take a tour and visit the help center for more information on this site and how it works. $\endgroup$
    – bob1
    Sep 24, 2023 at 20:41
  • 1
    $\begingroup$ To answer your question we would need to know a few more experimental details - have you performed titration of these antibodies to determine that they are working as expected? Have you performed any epitope blocking experiments for antibody specificity? Are you using a block of some sort in your staining? Is the 1ry suitable for PFA fixed samples? Is the 1ry antibody suitable for native protein? What controls are you using in your experiments? You should have primary and secondary antibody controls in each plate you run. Why are you using 2 fixatives? $\endgroup$
    – bob1
    Sep 24, 2023 at 20:45
  • $\begingroup$ Hello! Let me try answering your questions: 1. No I havent performed any such titration but my supervisor has and it has worked before for her. She has also said blocking is not necessary. since these antibodies should be specific enough to the virus in question, but what specific type of blocking were you thinking of? I dont think a block is used in the staining, since the primary antobody is specific for a viral protein of interest, and the secondary antibody is specific to the primary. $\endgroup$
    – Lina2
    Sep 29, 2023 at 20:03
  • $\begingroup$ 2. What do you mean by "1ry"? Primary antibody? And as in "suitable for native protein", are you referring to unspecific binding to other proteins of the cells? They antibody used should be specific enough to the virus Ive been told. The negative controls used are just wells with seeded cells, that have been treated the same, but not infected with any virus. Its the same cells, treated with both the primary and secondary AB. Do you mean I should have controls that have ONLY been treated with the primary OR the secondary AB, respectively? Too see if the 2dry are specific enough for the 1ary? $\endgroup$
    – Lina2
    Sep 29, 2023 at 20:07
  • $\begingroup$ 1) good, so long as you are using her conditions exactly as she did, this should be fine. Block is used to block non-specific binding of antibodies. It is very very very common that they can bind non-specifically, depending on the conditions used. Secondaries less commonly bind non-specifically. You should have appropriate controls in each experiment you perform to ensure that you can detect these problems if they occur. $\endgroup$
    – bob1
    Sep 29, 2023 at 23:19


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