Quantum dots thermal stability improves simultaneous phenotype-specific telomere length measurement by FISH-flow cytometry

Abstract

Telomere length analysis has been greatly simplified by the quantitative flow cytometry technique FISH-flow. In this method, a fluorescein-labeled synthetic oligonucleotide complementary to the telomere terminal repeat sequence is hybridized to the telomere sequence and the resulting fluorescence measured by flow cytometry. This technique has supplanted the traditional laborious Southern blot telomere length measurement techniques in many laboratories, and allows single cell analysis of telomere length in high-throughput sample formats. Nevertheless, the harsh conditions required for telomere probe annealing (82 degrees C) has made it difficult to successfully combine this technique with simultaneous immunolabeling. Most traditional organic fluorescent probes (i.e. fluorescein, phycoerythrin, etc.) have limited thermal stability and do not survive the high temperature annealing process, despite efforts to covalently crosslink the antigen-antibody-fluorophore complex. This loss of probe fluorescence has made it difficult to measure FISH-flow in complex lymphocyte populations, and has generally forced investigators to use fluorescent-activated cell sorting to pre-separate their populations, a laborious technique that requires prohibitively large numbers of cells. In this study, we have substituted quantum dots (nanoparticles) for traditional fluorophores in FISH-flow. Quantum dots were demonstrated to possess much greater thermal stability than traditional low molecular weight and phycobiliprotein fluorophores. Quantum dot antibody conjugates directed against monocyte and T cell antigens were found to retain most of their fluorescence following the high temperature annealing step, allowing simultaneous fluorescent immunophenotyping and telomere length measurement. Since quantum dots have very narrow emission bandwidths, we were able to analyze multiple quantum dot antibody conjugates (Qdot 605, 655 and 705) simultaneously with FISH-flow measurement to assess the age-associated decline in telomere length in both human monocytes and T cell subsets. With quantum dot immunolabeling, the mean decrease rate in telomere length for CD4+ cells was calculated at 41.8 bp/year, very close to previously reported values using traditional flow-FISH and Southern blotting. This modification to the traditional flow-FISH technique should therefore allow simultaneous fluorescent immunophenotyping and telomere length measurement, permitting complex cell subset-specific analysis in small numbers of cells without the requirement for prior cell sorting.

Figure 1

Protocol for the flow cytometry analysis of the telomere length. The first dot plot represents the initial gating of the cell population P1 to reject particles and aggregates. The second dot plot was used for the gating of the MNCs and the internal reference cells CT. The third dot plot represents the separation of the CD3+ cells from the CD14+ cells or monocytes within the gated MNCs. The next dot plot shows the gated CD3+CD4+ and CD3+CD4- cell populations from the previously gated CD3+ cell population. The PNA probes with and without FITC were then analyzed. The PNA FITC channels were then calculated for the gated CD14+, CD3+CD4+, CD3+CD4- and the gated CT cell populations.

Figure 2

(A) FITC histograms of MESF beads mixtures – M1 peak corresponds to the unlabelled MESF beads and M2 to M5 peaks correspond to the FITC labeled beads with known MESF values; (B) Linear regression of the measured FITC channel numbers versus known MESF values for the MESF beads reported in (A).

Figure 3

Loss of human CD3 labeling of human MNCs following heat treatment, using QD655 (top histogram), Alexa Fluor 647 (middle histogram) or phycoerythrin (bottom histogram). Samples were either heated at 82°C for 11 minutes (unfilled histograms) or not heated (filled grey histograms).

Figure 4

a: FITC PNA probe fluorescence for CD14+ monocytes, CD3+CD4+ and CD3+CD4-T cells (top row), calf thymocytes, normal donor MCs and Jurkat T cells (bottom row). b: Telomere length as a function of the age of the healthy donors for the different cell populations: CD3+CD4-, CD3+CD4+ and CD14+ cells. The linear regression of the telomere length vs. age is reported for the different cell populations.

Figure 4

a: FITC PNA probe fluorescence for CD14+ monocytes, CD3+CD4+ and CD3+CD4-T cells (top row), calf thymocytes, normal donor MCs and Jurkat T cells (bottom row). b: Telomere length as a function of the age of the healthy donors for the different cell populations: CD3+CD4-, CD3+CD4+ and CD14+ cells. The linear regression of the telomere length vs. age is reported for the different cell populations.