Cell barcoding minimizes the technical fluctuations in multiplex technologies. Especially, fluorescent cell barcoding is a standard method used in flow cytometry analysis for multiplexing. Similarly, mass-tag barcoding is used in mass cytometry for cell labeling. However, currently used cell barcoding reagents are unsuitable for labeling the live cells in long-term cell cultures.
Study: Hybrid Fluorescent Mass-Tag Nanotrackers as Universal Reagents for Long-Term Live-Cell Barcoding. Iamge Credit: Anusorn Nakdee/Shutterstock.com
In an article published in the journal Analytical Chemistry, palladium-based hybrid-tag nanotrackers with fluorescence activity were developed for live cell barcoding for mass and flow cytometry dual-modal readout. Furthermore, the preparation of nanotrackers, efficiency for cell internalization, physicochemical characterization, durability in long-term live cells, and compatibility of the nanotrackers with standardized cytometry protocols and reagents were demonstrated through the present study.
Nanotrackers were validated in drug response assays using long-term cell cultures with two barcoded cell lines. The present method is widely applicable for mass-tag and fluorescent barcoding without affecting the protein expression levels.
Reagents Used in Cell Barcoding
Cell barcoding is a labeling technique in which individual cells are labeled with unique nucleic acid sequences or barcodes to track them through space and time. Cell barcoding can be used to track millions of cells simultaneously and thus is an efficient approach for investigating heterogeneous populations of cells.
Cell barcoding enhances the sample consistency used in multiplex technologies. This consistency minimizes the technical variations arising due to the sample cross-contamination through loading loop, antibody staining, and variations in machine sensitivity.
While fluorescent cell barcoding allows multiplexing in flow cytometry analysis, mass cytometry enables efficient cell barcoding and multiplexing by implementing alternative methods. Furthermore, using metal isotopes tagged antibodies in mass cytometry facilitates the measurement of about 60 parameters per single cell and reveals unrecognized details on the heterogeneous cell populations.
Although lanthanides and commercially available palladium isotopes were used as first and second-generation mass cytometry barcodes, these metal-based barcoding reagents were intracellular labels and required permeabilization and fixation. Hence, these reagents were unsuitable for long-term culture assays. Despite the efforts invested in developing dual reagents, no focus on mass cytometry dual-modal readout in multiplexed drug studies and cell assays was aligned.
To this end, the narrow size of nanoparticles with uniform metal-loading capacity makes them promising analytes for mass cytometry. Moreover, polymeric nanoparticles are useful for tracking cellular proliferation, imaging, biosensing, and cellular proteomics.
Fluorescent Mass-Tag Nanotrackers for Cell Barcoding
In the present study, palladium-based nanotrackers with fluorescence activity were reported as hybrid-tag reagents for cell barcoding in long-term cell cultures. The chemical synthesis and characterization of the proposed nanotrackers or cell barcoding reagents were described.
Furthermore, the barcoding reagent’s compatibility with flow cytometry and standard mass reagents, and its performance in the biological systems were evaluated. The barcoding reagent’s compatibility for heterogeneous cell populations was also evaluated and combined with the dual application for flow and mass cytometry readout.
The proof of the proposed concept was performed with two hybrid-tag nanotrackers used for cell barcoding in two different cell cultures which were cocultured. Here, the barcoded cocultured cell cultures consisting of two different cell lines were monitored for their biological response when exposed to the drug long-term.
The results revealed that the hybrid-tag nanotrackers were versatile and robust live-cell barcoding reagents with non-toxicity. Furthermore, the two hybrid-tag nanotrackers were compatible with protein characterization and were suitable tools for longitudinal cell assays and multiplexed drug studies.
The single cells proteomic profiling was achieved by using a panel with 14 antibodies in the presence of the hybrid-tag nanotrackers. These nanotrackers provided robust live cell barcoding, which was compatible with metal-labeled antibodies, DNA intercalators, and cisplatin-based mass cytometry reagents, allowing the cell barcoding of heterogeneous cell populations along with mass and flow cytometry readout.
To conclude, mass hybrid-tag nanotrackers with fluorescent activity were developed as a new reagent for cell barcoding applications in long-term live cells and multiplexed drug assays, detected by mass and flow cytometry. These reagents showed advantages, including mass dual-modality, the durability of up to 14 days, fluorescence, universality, and stability for the long term. Although only two hybrid-tag nanotrackers were used in this barcoding strategy, it could be extended using other metal isotopes as tags.
The developed cell barcoding approach had no effect due to proteomic changes induced after exogenous perturbations. Parallel measurements of the cocultured cell line with nanotrackers-based cell barcoding reagents followed by treatment with doxorubicin were performed successfully.
The hybrid-tag nanotrackers were advantageous over other barcoding reagents since they are devoid of harsh conditions and can be used for drug-sensitive and minority heterogeneous cell populations used in cocultures. The present work was the first mass cytometry and dual fluorescent barcoding reagent with multiplexed drug assays and long-term live-cell barcoding.
Delgado-Gonzalez, A., Laz-Ruiz, J. A., Cano-Cortes, M. V., Huang, Y. W., Gonzalez, V. D., Diaz-Mochon, J. J., Fantl, WJ. et al (2022). Hybrid Fluorescent Mass-Tag Nanotrackers as Universal Reagents for Long-Term Live-Cell Barcoding. Analytical Chemistry. https://doi.org/10.1021/acs.analchem.2c00795