Immunology Lab, University Hospital, Saint-Etienne, France.
Faculty of Medicine of Tunis, University of El Manar, Tunis, Tunisia.
Cytometry A. 2023 Sep;103(9):732-735. doi: 10.1002/cyto.a.24780. Epub 2023 Aug 8.
Multiple immunolabeling introduces high risks of interferences between fluorescences. As an example, in analyzing T cell clonality, we recently reported a fluorescence resonance energy transfer (FRET) effect providing an unexpected signal on B770 (PE-Cy7) detector, on the Vβ-PE positive CD3 APC-Alexa750+ T cell subsets. Here, we report another FRET effect produced by the violet laser in Vβ-FITC positive CD3-Pacific Blue (PB) T cells providing signal on V550 (Krome Orange; KrO) detector. The study was performed on fresh whole blood, labeled with anti-CD3-PB, CD8-KrO, Vbeta FITC, Vbeta PE, CD4 AA750 then fixed, treated for erythrolysis, and washed before analysis on DxFlex cytometer from Beckman Coulter. Data were analyzed using Kaluza software. Using this panel, we repeatedly observed an added CD8dim-KrO (V550) cell population on all Vβ FITC positive T cells. The unexpected green signal excited by the violet laser was still observed after removing anti-CD8-KrO (FMO) but disappeared where either anti-CD3-PB or anti-Vβ-FITC was removed. The effect was also observed with an anti-TCR gamma delta-FITC labeling, but not with another FITC labeled antibody targeting a protein out of the CD3-TCR complex. The analysis of fluorochrome spectra confirms that PB emission and FITC excitation spectra partly overlap. This observation clearly reminds users that FRET can give misleading results in case of labeling of very close markers with complementary fluorochromes. This risk has to be considered in panel design. These observations clearly highlight the potential for FRET to give misleading results in cases where very close markers are labeled with complementary fluorochromes. This risk must be considered when designing panels. To our knowledge, this is the first description of a FRET between PB and FITC as acceptor thus excited by the violet laser.
多重免疫标记会带来荧光之间相互干扰的高风险。例如，在分析 T 细胞克隆性时，我们最近报道了一种荧光共振能量转移（FRET）效应，即在 B770（PE-Cy7）探测器上产生了一个意想不到的信号，而在 Vβ-PE 阳性的 CD3 APC-Alexa750+ T 细胞亚群上。在这里，我们报告了另一种 FRET 效应，它是由紫激光在 Vβ-FITC 阳性的 CD3-Pacific Blue（PB）T 细胞中产生的，在 V550（Krome Orange；KrO）探测器上提供信号。该研究是在新鲜的全血上进行的，全血用抗 CD3-PB、CD8-KrO、Vbeta FITC、Vbeta PE、CD4 AA750 进行标记，然后固定、进行红细胞溶解处理，再在贝克曼库尔特的 DxFlex 流式细胞仪上进行分析。数据使用 Kaluza 软件进行分析。使用这个面板，我们在所有 Vβ FITC 阳性 T 细胞上反复观察到一个额外的 CD8dim-KrO（V550）细胞群。在去除抗 CD8-KrO（FMO）后，仍然观察到由紫激光激发的意外的绿色信号，但在去除抗 CD3-PB 或抗 Vβ-FITC 时，该信号消失。这种效应也在用抗 TCR gamma delta-FITC 标记时观察到，但在用另一种不针对 CD3-TCR 复合物内蛋白的 FITC 标记抗体时观察不到。荧光染料光谱分析证实，PB 发射和 FITC 激发光谱部分重叠。这一观察结果清楚地提醒用户，在使用互补荧光染料标记非常接近的标记物时，FRET 可能会产生误导性结果。在面板设计时必须考虑到这种风险。这些观察结果清楚地强调了在使用互补荧光染料标记非常接近的标记物时，FRET 可能会产生误导结果的可能性。在设计面板时必须考虑到这种风险。据我们所知，这是 PB 和 FITC 之间作为供体的 FRET 的首次描述，因此被紫激光激发。
