Journal: Chemical & Biomedical Imaging

Article Title: Monitoring Macrophage Polarization with Gene Expression Reporters and Bioluminescence Phasor Analysis

doi: 10.1021/cbmi.4c00049

Figure Lengend Snippet: Macrophage polarization reporters together with spectral phasor analysis enable single-cell polarization state readout. (A) Cartoon depiction of BRET reporter expression dependent on promoters upregulated in either M1-polarized ( NOS2 promoter-CeNL, top) or M2-polarized ( STAT6 promoter-YeNL, bottom) macrophages. (B) Cartoon depiction of changes in bioluminescence emission color of cells stably expressing the M1 (top) or M2 (bottom) BRET reporter construct upon stimulation with LPS or IL-4 + IL-13, respectively. (C) Emission spectrum (left) and position in the spectral phasor plot (right) of the M1 BRET reporter (CeNL, top) and M2 BRET reporter (YeNL, bottom).

Article Snippet: The NOS2 promoter region was amplified from the pGL2-NOS2Promoter-Luciferase plasmid (Addgene no. 19296).

Techniques: Expressing, Stable Transfection, Construct

Journal: Chemical & Biomedical Imaging

Article Title: Monitoring Macrophage Polarization with Gene Expression Reporters and Bioluminescence Phasor Analysis

doi: 10.1021/cbmi.4c00049

Figure Lengend Snippet: Stimulation with different activators allowed validation of gene expression reporter cell lines via bioluminescence phasors. Cells stably expressing polarization gene expression reporters: NOS2 -CeNL (A, B) or STAT6 -YeNL (D, E). Cells were stimulated either toward M1 (A, D, IL-4 20 ng/mL and IL-13 20 ng/mL) or M2 (B, E, LPS 5 μg/mL) polarization. For each condition (A, B, D, E) we are reporting, left to right, a cartoon depiction of the cell status upon stimulation, cell segmentation masks, bioluminescence intensity images, false-colored images based on the pixelwise phasor position, phasor plot reporting the median phasor position in single cells, and phasor plot of the pixelwise phasor distribution for the whole field of view. Scale bar is 30 μm. Single cell intensity quantification is reported for NOS2 -CeNL (C) and STAT6 -YeNL (F) cell lines. Data are represented by box and whiskers plots. The box represents the median (solid line) and standard deviation. The whiskers go down to the smallest value and up to the largest. All the points are shown. One-way ANOVA (K–W test), p > 0.05 (ns), 0.05 > p > 0.01 (∗), 0.01 > p > 0.001 (∗∗), 0.001 > p > 0.0001 (∗∗∗), p < 0.0001 (∗∗∗∗). For each condition, a minimum of two technical replicates were collected.

Article Snippet: The NOS2 promoter region was amplified from the pGL2-NOS2Promoter-Luciferase plasmid (Addgene no. 19296).

Techniques: Biomarker Discovery, Gene Expression, Stable Transfection, Expressing, Standard Deviation

Journal: Chemical & Biomedical Imaging

Article Title: Monitoring Macrophage Polarization with Gene Expression Reporters and Bioluminescence Phasor Analysis

doi: 10.1021/cbmi.4c00049

Figure Lengend Snippet: Imaging macrophage reporter cells in 3D tissue mimic. (A) Cartoon depiction of a mixed cell population embedded in a 3D collagen matrix. Luciferase substate (furimazine) was added from the top and detection is collected from the bottom of the sample. Measurements were taken at three different depths (top, middle, bottom) as described under . Quantification of bioluminescence intensity of the NOS2 -CeNL reporter when stimulated toward M1 (B) and of the STAT6 -YeNL reporter when stimulated toward M2 (C). Measurements were taken at different time points after incubation with cytokines (4, 12, and 16 h) and at different depths. Data are represented by box and whiskers plots. The box represents the median (solid line) and standard deviation. The whiskers go down to the smallest value and up to the largest. All the points are shown. One-way ANOVA (K–W test), p > 0.05 (ns), 0.05 > p > 0.01 (∗), 0.01 > p > 0.001 (∗∗), 0.001 > p > 0.0001 (∗∗∗), p < 0.0001 (∗∗∗∗). Scatter plots of the bioluminescence intensity as a function of the spectral phase (in radians) of the cells imaged after 16 h of cytokine incubation for M1 (D) and M2 (G) stimulation. The M1-polarized NOS2 -CeNL cell population is highlighted by the blue oval in (D), and the M2-polarized STAT6 -YeNL cell population is highlighted by the yellow oval in (G). Bioluminescence intensity (left) and phase-colored images (right) of the “top” depth of a mixed cell population after 16 h of stimulation toward M1 (E) or M2 (H). Scale bar is 60 μm. Average phasor location of single cells (left) and of the whole field of view (right) of the mixed cell population stimulated toward M1 (F) or M2 (I), corresponding to the field of views shown in (E) and (H), respectively. For each condition, a minimum of two biological replicates (each with two technical replicates) were collected.

Article Snippet: The NOS2 promoter region was amplified from the pGL2-NOS2Promoter-Luciferase plasmid (Addgene no. 19296).

Techniques: Imaging, Luciferase, Incubation, Standard Deviation

Journal: Chemical & Biomedical Imaging

Article Title: Monitoring Macrophage Polarization with Gene Expression Reporters and Bioluminescence Phasor Analysis

doi: 10.1021/cbmi.4c00049

Figure Lengend Snippet: Primers Used for Amplification of the Promoter Regions and Gene Inserts a

Article Snippet: The NOS2 promoter region was amplified from the pGL2-NOS2Promoter-Luciferase plasmid (Addgene no. 19296).

Techniques: Amplification