Abstract
Measurements of single-cell proteins provide key insights in studies related to cellular heterogeneities, while the absolute quantification of single-cell non-surface proteins remains elusive. This paper presents a droplet-based microfluidic flow cytometry enabling high-throughput quantification of both surface and intracellular proteins of single cell. Gradient solutions of fluorescently labeled proteins were first flushed into constriction channels with fluorescence signals detected to obtain calibration curves. Then, droplets encapsulating single cells stained with fluorescently labeled proteins were aspirated into constriction channels with excited fluorescence signals detected and translated into numbers of proteins based on the calibration curves. Based on this approach, both suspended and adherent cells with both surface and intracellular staining were processed. More specifically, the numbers of ConA binding sites of K562 cells, anti-β-actin antibody binding sites of A549 and HeLa cells were quantified as 3.43 ± 4.07 × 106/cell (Nc = 530); 9.20 ± 5.21 × 105/cell (Nc = 1073); and 1.44 ± 0.75 × 106/cell (Nc = 1138), respectively. As a high-throughput microfluidic platform, this technique can add a quantitative approach to measure single-cell proteins with arbitrary distributions within cells.
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The code used during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
The authors would like to acknowledge financial supports from the National Natural Science Foundation of China (Grant No. 61825107, 61922079, 62001042); Key Project (QYZDB-SSW-JSC011), Instrument Development GJJSTD20210004), Youth Innovation Promotion Association and Interdisciplinary Innovation Team of Chinese Academy of Sciences.
Funding
Financial supports from the National Natural Science Foundation of China (Grant No. 61825107, 61922079, 62001042); Key Project (QYZDB-SSW-JSC011), Instrument Development (GJJSTD20210004), Youth Innovation Promotion Association and Interdisciplinary Innovation Team of Chinese Academy of Sciences.
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Conceptualization: JC, JW, HY; data curation: HY, YW, BF; methodology: HY, YW, BF, TZ; formal analysis and investigation: HY, YW, BF, LL, TZ; writing—original draft preparation: HY; writing—review and editing: JC, JW; funding acquisition: JW, DC; resources: YW, LL, TZ; software, validation and visualization: DC, LL, TZ; supervision and project administration: JW, JC, DC.
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Yang, H., Wei, Y., Fan, B. et al. A droplet-based microfluidic flow cytometry enabling absolute quantification of single-cell proteins leveraging constriction channel. Microfluid Nanofluid 25, 30 (2021). https://doi.org/10.1007/s10404-021-02427-w
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DOI: https://doi.org/10.1007/s10404-021-02427-w