Human-T-Cell-Selective Fluorescent Probe

Abstract

The identification of T and B lymphocytes has relied on using antibodies against different biomarkers as the gold standard. Emerging small molecule-based fluorescent probes have the potential to replace antibodies. Herein, we report the first human-T-cell-selective fluorescent probe, Mito thermo yellow (MTY), achieving the live T cells' distinction from B cells, which was previously impossible without the help of antibodies. The unexpected cell selectivity of MTY is attributed to the higher mitochondria mass and membrane potential of T cells over B cells. This study enriches the toolbox for live cell distinction from complex cell communities.

Keywords: cell recognition; fluorescent probes; high-throughput screening; mitochondria.

Figure 1

Development progress of T- and B-cell identification.

Figure 2

Selectivity of MTY in human T and B cells. (a) MTY performance in the lymphocyte of blood. The WBCs were incubated with MTY (1 µM) for 1 h. (b,c) Selectivity in isolated human T and B cells. T cells were colocalized with MTY and CD3 antibody. B cells were colocalized with MTY and CD19 antibody. Fluorescence images (b) and flow cytometry (c) were taken after 1 h incubation with MTY (100 nM) and 30 min with antibody. (d) Quantitative comparison of fluorescent intensity by flow cytometry. Fluorescent intensity in Figure 3c was normalized and represented as histogram. (e) Selectivity of MTY in mixed T and B cells. T cells and B cells were randomly mixed, and then treated with MTY (100 nM), Hoechst (1 µg/mL), CD3 antibody and CD19 antibody.

Figure 3

Localization of MTY. (a) Localization of MTY in human primary T cells. T cells were treated with MTY (100 nM), Mitotracker Green (200 nM), Cellmask Deep Red (5 ug/mL) and Hoechst (1 µg/mL) for 1 h, 30 min, 30 min and 10 min, respectively. (b,c) Selectivity of MTY in the H9 and Daudi cells. Fluorescence images (b) and flow cytometry (c) were obtained after 1 h incubation with MTY (100 nM). Fluorescent intensity evaluated by flow cytometry was normalized and represented as histogram. (d) Localization of MTY in H9 cells. H9 cells were treated with MTY (100 nM), Mitotracker Green (200 nM) and Hoechst33342 (1 µg/mL) for 1 h, 30 min and 10 min, respectively. (e) Display of the colocalization areas of the red and green channels in (d).

Figure 4

Mechanism for the selectivity of MTY. (a,b) MMP effect on the fluorescence of MTY in human T and B cells. The cells were first treated with CCCP (50 µM) for 30 min, and then MTY (100 nM) was added for 1 h. Fluorescent intensity evaluated by flow cytometry was normalized and represented as histogram. (c,d) The fluorescence of MTG (200 uM) and Rhod123 (100 uM) in human T and B cells. The fluorescence images were taken after 30 min incubation of MTG and Rhod123. Fluorescent intensity evaluated by flow cytometry was normalized and represented as histogram. (e) The proposed staining mechanism of MTY.