Authors: I. Sotnikov, S. Jaron, M. Konopleva, S. O’Brien, M. Andreeff, J. Hildebrant, J. Manis, J. Brown, I. Vorobjev, N. Barteneva
Affilation: Immune Disease Institute, Harvard Medical School, United States
Pages: 424 - 427
Keywords: intracellular flow, qdot nanocrystals, zap-70
We present, for the first time, the methodological approach for the measurement of cytoplasmic Zap-70 and nuclear Ki-67 proteins with Qdot® nanocrystals-labeled antibodies. Mononuclear cells from chronic lymphoid leukemia patients were used to detect Zap-70, and Jurkat T-cells for Ki-67. Intracellular staining kits based on saponin and paraformaldehyde are optimal for the delivery of Qdot® nanocrystals inside cells. The single staining with Qdot® 565, 605, or 655 labeled antibodies is operative in intracellular flow. In multicolor panels, the most robust results are obtained with the Qdot® 655 detected in the APC channel (633 nm excitation, 660/20 nm band pass or customized 655/20 filter) to simplify the spectral overlap compensation. In 3-, 4-, and 5-color combinations signal brightness for intracellular Qdot® 655 does not decrease, whereas positive to negative signal ratio is higher compared to organic dyes. The optimized combination of fluorophores for 5-color panel includes APC-Cy7, PE, PE-Cy7, FITC, and Qdot® 655. We used CD3, CD19, CD38, CD5 surface markers. Intracellular Qdots® outperformed organic fluorophores probed in the same samples. Qdot® nanocrystals are a robust tool in intracellular flow cytometry due to the narrow emission spectrum, brightness and stability of this fluorophore.
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