We ran a flow cytometry experiment where Fc blockers were added into the antibody cocktail. Other antibodies added include:
1. anti-CD3-PE-Cy7
2. anti-CD4-APC
3. anti-CD8-AmCyan
4. anti-CD14-APC-Cy7
5. anti-CD19-BV605
6. anti-pSTAT1-AF488
7. anti-pTAT3-PE
8. anti-pSTAT5-BV421
Before Fc blockers were added, the monocyte population expressed high levels of CD3. We speculated that the anti-CD3 antibody might undergo non-specific binding to FcR. However, adding Fc blockers did not change the CD3 expression.
Is there any possible scenario of why this happened?
Is there any chance that you isolated T-Cell/monocyte complexes? The T-cells would express CD3.
Hi Kezia Singgih
please first explain more about your experiment. how do you sure, your population is monocytes or how are your gating strategies?
However, recently some publication claimed that time monocyte can differentiate to T cell and express CD3 (Article Circulating T cell-monocyte complexes are markers of immune ...
,Article CD3+ Macrophages Deliver Proinflammatory Cytokines by a CD3-...
).
I recall a former colleague telling me about monocytes exhibiting non-specific staining and that they thought it was directly related to a fluorochrome (tandem dye). I just ran a quick search and came up with this reference.
https://www.jimmunol.org/content/198/1_Supplement/81.25
Dear Kazia,
Before thinking of monocyte+lymphocyte complexes, I suggest you to look up for below issues:
1. Check all of your solutions, washing/staining buffers need to be fresh, their pH should be 7.2-7.4 . Sometimes crystallization can be the cause of false staining
2. If you have not titrated your antibodies, perform titration analysis for all of your antibodies.
3. FMO control is important, so especially for CD3 make a FMO control. FMO = Fluorescence Minus One. This will show you if there is any spill from other colors.
Shortly, validate your panel for all aspects to find the reason
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