Spiking means mixing a volume of unstained cells with stained cells to get a negative peak where all the population is positive for some marker (like we spike for CD45 in peripheral blood, as all the cells are positive for cd45).

If your whole population is expressing YFP, which you are going to observe through GFP tagged antibody, you would not find any negative population.

To see the negative population spike your stained tube with unstained population. You will get both positive and negative peak.

If you have pure population, then you would not see negative population. For that you need to Spike the stained tube (GFP tube) with control cells (which doesn't express GFP.

Looks like all your cells are expressing YFP. You likely will be able to see YFP expression on your cells without staining with anti-GFP antibodies as YFP and GFP emission maximums are very close 527nm (YFP) and 509nm (GFP). That's why your "unstained " (but actually YFP positive) cells give shift in GFP channel fluorescence.

I agree with Natallia Mikhalkevich 's comment. If your YFP has not been compromised by fixing or other manipulations you don't need to take the d-tour via AB labeling. Your cells will emit YF upon excitation.

Anyway, it's important to have a negative control as well, so spiking is, as suggested above, definitely recommended.

Good luck!

Thank you all for your responses. Due to the total shift in expression, I analysed the MFI instead. For the GFP antibody control, i used the isotype of the GFP antibody, and the MFI is low. However, when I compare my yfp-tagged mouse to a wild-type mouse, both had almost the same MFI values, which are higher than the isotype control. This is the part that I find it very strange. Does this mean the GFP antibody is not specific or it created more noise? Or I didn't stain enough (stained for 1hr) ?

Kanika Arora Jitendra Kumar Shandilya May I ask what do you mean by spiking?

Natallia Mikhalkevich To begin with, the YFP expression is almost negligible in the mouse. Hence, I used the GFP antibody in hopes to enhance the signal. Also, to avoid the possible contribution of the "unstained" YFP expressing cells, I used the PE-anti GFP in the PE channel instead. So technically, all stained GFP cells should be in PE.

The MFI values are isotype 126, wildtype 2566, YFP-tagged mice are 2515, 2740, 2718.

Hi Samantha. Just couple of questions:

Do you have histogram (for PE channel) of unstained wild type (no YFP -expressing) cells?

Do you have surface or intracellular YFP -coupled protein expression? What cells were used for isotype control?

Were Isotype control antibodies provided by the same company that provided anti-GFP antibodies with the same fluorochrome: protein (F/P) ratio? If not, don't rely on this isotype control, it will only cause a confusion. In this case there is likely nonspecific staining with your anti-GFP antibodies or you don't have any staining at all (histogram (PE channel) of unstained wt cells can help to answer this question).

You can do titrations to find the optimal antibody concentration (significantly reduce antibody concentration), do some extra washes, perform Fc block prior to staining in case you have Fc-receptor expressing cells (B cells, macrophages, NK cells, etc) to get rid of nonspecific staining.

Do you have an opportunity to look at your "unstained" cells from YFP-expressing and wild type mice in FITC channel of fluorescent microscope?

Do your histograms represent only viable cells (dead cells and aggregates are excluded from analysis)?

PE-channel fluorescence without proper compensation will still be affected by YFP-fluorescence of "unstained" YFP expressing cells.

Natallia Mikhalkevich

• No histogram (for PE channel) of unstained wild type cells.
• Intracellular YFP-coupled protein expression. YFP-expressing cells were used as isotype control.
• GFP antibody usage was 5ul/test. Hence, I used 1ul of the isotype per sample. I do not know the protein (F/P) ratio.
• I have done extra washes, Fc block prior to staining. I will do more washes.
• I will do the titration on the optimal antibody concentration!
• I have done imaging on the YFP-expressing and wild type mice in FITC channel of fluorescent microscope through cryosections. The signal is really poor, hence when I used anti-gfp antibody, I could finally detect my YFP-expressing cells, while the wildtype is clean.
• Do you think collagense digestion might affect the YFP expression? I have compared collagenase A and IV, but there is no difference.

Spiking means mixing a volume of unstained cells with stained cells to get a negative peak where all the population is positive for some marker (like we spike for CD45 in peripheral blood, as all the cells are positive for cd45).

For spiking please see 4th paragraph.

https://www.bio-rad-antibodies.com/flow-antibody-titration.html

Not sure that collagenase digestion is affecting YFP expression. Flow cytometry of cells derived from tissues is rather tricky. If your YFP signal is really weak, especially if it's intracellular (not surface) you might not be able to detect it with flow cytometry, especially if you have very different cell types, as background fluorescence from large granular cells and cell aggregates may mask weak YFP signal from cells with lower background fluorescence. Also many cell types in tissues are autofluorescent, this also affect tissue flow cytometry usage. Flow cytometry gives only distribution of cells with different levels of fluorescent intensity. Fluorescent microscopy would be the best choice in this case, though will not allow you to analyze many cells in a short period of time and combine multiple dyes.

If you have intracellular YFP expression, it makes sense to fix ( 2-4% buffered PFA (methanol free!!!), not methanol fixation for YFP/GFP, as methanol is a known GFP quencher) and permeabilize the cells (saponin will likely be the best choice), as PE molecules themselves in anti-GFP -PE antibodies are very large and don't go inside the cells easily during intracellular staining. PE has high stain index compare to other fluorescent dyes and is a good choice for dim markers, but can be problematic for intracellular staining due to large size. Also it make sense to sonicate the PE-antibodies before use, as PE tend to make aggregates itself.

For tissues with large amount of autofluorescent cells (like skin) it's better to use red/ far red dyes (APC, AF647, etc), they are not so bright as PE, but there is weaker autofluoirescent signal in red channels.

You can add fixable viability dyes for fixed cells as a first step staining to remove dead cells from consideration later, it will help you to get rid of nonspecific signal a lot, but their choice depends on laser/filters availability on the flow cytometer.

I used magnetic dead cell removal kits for tissue cells flow cytometry prior to staining and it helped a lot.

Natallia Mikhalkevich I have already conducted western blot to confirm the YFP. And with the imaging, I am convinced that the expression is present but low.

I have used foxp3 staining kit for intracellular staining and also included fixable viability dye. Hence, my histograms represent only viable cells (dead cells and aggregates are excluded from analysis).