An Elucidation of Neutrophil Functions against Mycobacterium tuberculosis Infection
Figure 4
Assay of MDA in neutrophil lysates at 24 hours after infection with H37Rv ( ). Free radical levels in neutrophil lysates were determined by measuring the levels of MDA using a colorimetric assay kit from Cayman. These elevated MDA concentrations are indicative of elevated free radical concentrations. Treatment with either NAC or L-GSH reduced the amount of MDA. (b) and (c): Cytokine measurements performed in supernatants derived from H37Rv-infected neutrophils. Levels of TNF-α (b) and IL-6 4(c) in neutrophil supernatants were measured by ELISA. (d): Determination of phagosome acidification by quantifying the colocalization of FITC-labeled H37Rv with lysotracker: neutrophils were stained with lysotracker and infected with FITC labeled H37Rv. Infected neutrophils were incubated for 24 hours on sterile glass cover slips in the presence and absence of GSH-enhancing agents. Neutrophils were fixed with 3.8% PFA in PBS for 30 minutes, and the coverslips were mounted on a clean glass slide using DAPI. Slides were then viewed using an inverted fluorescent microscope for the colocalization of FITC-labeled H37Rv with lysotracker which is indicative of phagosome acidification. Images were obtained using an integrated digital camera and analyzed using ImageJ. ; . Graph shows mean ± standard error.
