Journal: bioRxiv

Article Title: Design and Evolution of an Orthogonal HaloTag for Multiplexed Labeling in Cells

doi: 10.64898/2026.05.14.725131

Figure Lengend Snippet: a) Flow cytometry of HEK293T cells expressing either HaloTag-T20 or OrthoTag-H2B and labeled with different concentrations of bCA-TMR (left) or CA-BDY (right). Fluorescence was normalized to maximum fluorescence and background fluorescence for each experiment, and data are the averages and standard errors of the mean for three biological replicates. b) Representative confocal fluorescence microscopy image of HEK293T cells expressing both HaloTag-T20 and OrthoTag-H2B and labeled with 100 nM CA-BDY and 100 nM bCA-TMR for 1 h before washing, fixing, and staining with Hoechst 33342. c) Correlation coefficients for the Hoechst, TMR, and BDY channels from confocal fluorescence microscopy (n = 20 cells). The line shows the median value, boxes show the middle quartiles, and whiskers show the minimum and maximum values in each dataset. Coefficients range from -1 to 1, with -1 representing perfect anti-localization and 1 representing perfect colocalization. Coefficients were calculated using CellProfiler.

Article Snippet: Cells were washed three times with PBS again and incubated for 5 min at room temperature with 1 mL of PBS and 0.4 μL of 10 mg/mL Hoechst 33342 trihydrochloride trihydrate in water (Life Technologies).

Techniques: Flow Cytometry, Expressing, Labeling, Fluorescence, Microscopy, Staining