2.3. Biological application of TPEARG at the cellular level
Afterward, due to the outstanding detection performances of TPEARG toward arginase, its biological applications were investigated in living cells in detail. Research works reported that arginase was high expressed in the M2 macrophages[6a, 6b]. Thus, the RAW 264.7 cells were stimulated into M1 or M2 type using IFN-γ/LPS or IL-4, respectively. The RAW 264.7 cells treated with PBS (pH=7.2) and BEC (An arginase inhibitor) were set as the control groups. Then, the cells were incubated with 1 μM TPEARG and imaged under laser scanning confocal microscope. Compared to the undifferentiated RAW 264.7 and M1 macrophage, the M2 macrophage displayed over 20-fold fluorescence intensity enhancement (Figure 3A and B). In addition, when the arginase was inhibited by BEC, the cellular fluorescence intensity decreased significantly. These results suggest that TPEARG possess splendid potential in the detection of cellular arginase. To further explore the dynamic detection capability of TPEARG, we applied the TPEARG in M2 macrophages with sequential levels of arginase. M2 macrophages were pretreated with BEC for incremental time to obtain reduced expressions of arginase (Figure S5). Then, the TPEARG was used to observe the variation of cellular arginase. As displayed in Figure 3C and D, with the incubation time increased, the cellular fluorescence intensity decreased gradually. These results confirm that our established imaging method hold great potential in the dynamic detection of cellular arginase.