My dear community,
I am writing to seek your collective expertise regarding an unexpected result in a flow cytometry experiment that has left me puzzled. Despite thorough optimization and compensation, I am observing a dramatic and consistent discrepancy in T-cell subset quantification between the two antibody panels.
Experimental setup:
The Problem:
The issue arised when I analyzed the classical surface-based T-cell subsets (gated on live, single cells, CD3+CD4+/CD8+) before looking at any intracellular targets. In the panel 2 (non-fixed/non-permeabilized) cells, the central memory (CD45RO+CCR7+) T-cell population is dominant: CD4 - 84.02%; CD8 - 73.32%. However, in the panel 1 cells that underwent fixation/permeabilization, the distribution is drastically different: CD4 - 31.15%; CD8 - 9.61%. The majority of cells in this group now exhibit a CD45RO-CCR7+ phenotype (naive-like), which was a minor population in the CFSE+ sample.
This is a paired sample from the same donor, processed in parallel. The only difference is the initial CFSE label and the fact that the panel 1 cells were fixed/permeabilized.
My questions to the community:
I have double-checked my gating strategy and compensation, and the effect is too large to be attributed to those factors. Any insights, suggestions, or shared experiences would be immensely appreciated. I am happy to provide more specific details on antibodies clones and buffers used in a follow-up.
Thank you in advance for your help.
Hello,
The fixation can have an effect on the fluorescent conjugates, but not usually on the epitopes. Some fluorescent molecules can stop working properly as seen in your 1rst panel.
Louis-Charles Béland, thank you for your reply. My anti-CD45RO antibody is APC eFluor780 (Thermo #47-0457-42). They are not reported to be significantly vulnerable to cell fixation or permeabilization. But I will check this myself.
Don't hesitate to contact ThermoFisher tech support, they usually answer in 1 or 2 business day.
I tested the antibodies by adding them to AbC total compensation beads and then incubating them with the Fixation buffer and True Phos buffer that I use. The Fixation buffer had no effect on the beads' MFI even after 20 minutes of incubation. However, adding the True Phos buffer caused a decline in MFI; it was a 4-5 fold drop. The positive beads remained positive for APC eFluor780. I am not sure if this drop is enough for the memory subsets to disappear from the analysis.
Yes fixation can mask epitopes and prevent the antibody binding. There is a very specific biolegend CD8 antibody I have that does this and I have never managed to get it to work on fixed samples but it works beautifully on fresh ones. The first time I saw it the effect was so dramatic I though I'd forgot to put the antibody in the master mix and samples.
Samantha Le Sommer that's interesting! Yesterday I observed a 15 fold decrease in anti-CD8 BV510 fluorescence (BioLegend #301048) on my fixed/permeabelized CD8 T cells as apposed to the same cells when they were fresh. But I hope that was not an epitope problem. Will try to stain cells after they were permeabelized so that Fix and True Phos Perm Buffers won't hurt the dyes.
Yes, the fixation step can sometimes alter epitopes and therefore ligation of mAbs. This issue is variable to mAb clone to clone ("fixation sensible clones" vs insensible ones). Also, the permeabilization buffer impacts on FSC/SSC of the cells. I would try other buffers. In our hands the Dako IntraStain kit (ref K2311) has the least "cell size" impact.
Hope this helps, good luck in your experiments!