Nanotech 2010

Qdot® nanocrystals in intracellular flow: ZAP-70, KI-67 and five-colors fluorophores panel

I. Sotnikov, S. Jaron, M. Konopleva, S. O’Brien, M. Andreeff, J. Hildebrant, J. Manis, J. Brown, I. Vorobjev, N. Barteneva
IMMUNE DISEASE INSTITUTE, HARVARD MEDICAL SCHOOL, US

Keywords: intracellular flow, qdot nanocrystals, zap-70

Abstract:

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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