Tertov V V, Sayadyan H S, Kalantarov G F, Molotkovsky J G, Bergelson L D, Orekhov A N
Institute of Experimental Cardiology, U.S.S.R. Cardiology Research Center, Moscow.
J Immunol Methods. 1989 Mar 10;118(1):139-43. doi: 10.1016/0022-1759(89)90064-1.
Flow cytometry was used for the isolation of hybrid cells immediately after fusion. Precursor cells were stained by two lipophilic fluorescent probes: perylenoyl-labeled triglyceride (perylenoyl-TG, green fluorescence, 520 nm) and rhomdaminyl-labeled triglyceride (rhodaminyl-TG, red fluorescence, greater than 580 nm). Since the maximum emission of perylenoyl-TG coincides with the maximum absorbance of rhodaminyl-TG, the two fluorescent dyes form an effective donor-acceptor pair. Cells stained by perylenoyl-TG (0.25-1 microgram/ml) at the excitation wavelength of 457 nm displayed high intensity of fluorescence in the green region (520 nm), and low intensity of fluorescence in the red region (greater than 580 nm). Using the same conditions, cells that were stained by rhodaminyl-TG displayed a low intensity of fluorescence in both regions. When cells were simultaneously labeled by perylenoyl-TG and rhodaminyl-TG (used in a concentration ratio of 1:10, respectively) essentially total energy transfer was observed, and the cells exhibited a high intensity of red fluorescence. After the fusion of cells which had been separated stained by perylenoyl-TG and rhodaminyl-TG, the hybrid cells containing the two fluorescent probes had a high intensity of red fluorescence. Resonance exitation energy transfer between the two fluorescent dyes permits effective sorting of hybrid cells by flow cytofluorometry.
融合后立即使用流式细胞术分离杂交细胞。前体细胞用两种亲脂性荧光探针染色：苝酰基标记的甘油三酯（苝酰基 - TG，绿色荧光，520 nm）和罗丹明基标记的甘油三酯（罗丹明基 - TG，红色荧光，大于580 nm）。由于苝酰基 - TG的最大发射波长与罗丹明基 - TG的最大吸收波长重合，这两种荧光染料形成了有效的供体 - 受体对。用苝酰基 - TG（0.25 - 1微克/毫升）染色的细胞在457 nm激发波长下在绿色区域（520 nm）显示出高强度荧光，在红色区域（大于580 nm）显示出低强度荧光。在相同条件下，用罗丹明基 - TG染色的细胞在两个区域均显示出低强度荧光。当细胞同时用苝酰基 - TG和罗丹明基 - TG标记（浓度比分别为1:10）时，基本上观察到了完全的能量转移，细胞呈现出高强度的红色荧光。用苝酰基 - TG和罗丹明基 - TG分别染色后分离的细胞融合后，含有这两种荧光探针的杂交细胞具有高强度的红色荧光。两种荧光染料之间的共振激发能量转移允许通过流式细胞荧光术有效地分选杂交细胞。
