Based on the data obtained by two independent methods, which differ in i) probe delivery method (microporation of live cells vs. FISH with fixated cells) and ii) read-out (flow cytometry vs. fluorescence microscopy), it is likely that the different staining intensities are due to target engagement of the probes. However, the study also highlights difficulties. The fold changes of MFI induced by the probes were relatively small, probably due to the mild microporation protocol. Under these conditions, only a small fraction of the cells will receive probes. Harsher microporation would result in more efficient delivery. However, this increases the number of dead cells, which tend to acquire large amounts of probe and therefore give false positives. Considering the relatively weak staining, it remains difficult to discriminate against cells that have higher autofluorescence than the target cells. We speculate that improved delivery methods may help. Nevertheless, a key challenge remains: at endogenous expression levels of TCR mRNA only few of the probes may find the target and the majority of the probes may remain unbound. Despite this, in our studies we observed a few target cells that showed higher fluorescence than the non-target cells. Most likely, these highly fluorescent cells have higher levels of target mRNA due to cell-to-cell variability. Focusing the analysis on these cells should allow distinctions to be made based on TCR mRNA expression.