We had observed occasional, variable performance of QD staining that was eventually traced to certain staining conditions when using particular lots of fixative. As shown in , when cells were stained with CD8 QD655, followed by fixation in formalin, there was a complete loss of fluorescence when using lot “A” compared to the other lots. Lot “C” exhibited partial loss of fluorescence. Based on previous reports of heavy metal poisoning of QD luminescence (4
), we reasoned that addition of EDTA during the fixation might reverse the inhibition. As shown in , this was the case; the fluorescence resulting after fixation in the presence of 1 mM EDTA could completely protect the QD fluorescence. Notably, addition of EDTA alone, in the absence of fixative, marginally reduced QD fluorescence.
Variable quenching of QD fluorescence by different lots of fixative
As shown in , the use of different staining media in the absence of fixation did not affect QD fluorescence. However, after adding 1% formalin from lot “A” we observed a dramatic loss of QD fluorescence in the samples stained with PBS or RPMI but not with the R10 medium. This suggests that the contaminant in the formalin quenched the QD fluorescence in a manner that was inhibited by the additional serum present in R10 medium; certainly, there could be sufficient metal-binding proteins in serum to chelate the contaminant. Finally, as shown in , addition of 1 mM EDTA could inhibit the effect of the contaminant in the formalin – but only if the EDTA was present concurrent with the formalin. Adding EDTA subsequent to the formalin did not reverse the inhibition of fluorescence. These data show that the contaminant was likely a chelatable heavy metal cation that, once intercalated into the QD, could not be removed. Finally, illustrates that the elimination of fluorescence occurs equally well with another QD reagent, QD605.
Different media can alter the QD fluorescence quenching by the contaminated fixative
In order to determine which heavy metals might be the source of the inhibitory activity, mass spectrometric analysis of the different lots of fixative were performed to quantify a number of possible cations. As shown in , the analysis revealed measurable levels of copper and iron present in lot “A”, suggesting that one of these is likely the root cause.
Mass spectrographic quantification of total copper and iron in staining solutions
To determine the precise effect of various metal cations, compensation beads were stained with CD8 QD655 and treated with various levels of several heavy metal cations: Zn+2, Fe+2, Fe+3 and Cu+2 (). As shown, all but ferrous ions could inhibit fluorescence at some concentration below 1 mM, although ferrous and zinc ions inhibited at much higher concentrations, i.e., 0.1 and 0.5 mM respectively. Notably, cupric ions showed a half-maximal inhibitory concentration that is very low, 20 nM.
Concentration-dependent inhibition of QD fluorescence by heavy metal cations
In other experiments, we found that 30 µM cupric ions can eliminate the fluorescence of at least six different QDs, including QD545, QD565, QD605, QD655, QD705, and QD800 (data not shown).
Finally, we demonstrate that this property of QD can be used to re-stain cells with different reagents of the same fluorescence. In , fluorescence distributions are shown for PBMC that were stained with QD655 conjugated to anti-CD4 or anti-CD8. After treatment with cupric sulfate, the cells are rendered completely non-fluorescent for the QD reagents (with the antibodies presumably still bound). After thorough washing with 1 mM EDTA, the cells were successfully re-stained with QD655 conjugated to the other antibody. We observed slightly less staining in the second step compared to the first step, which may be attributable to incomplete removal of the cupric sulfate.
Potential utility of the elimination of fluorescence