Journal: Frontiers in Immunology
Article Title: Trypanosoma cruzi Infection Induces Cellular Stress Response and Senescence-Like Phenotype in Murine Fibroblasts
Figure Lengend Snippet: Trypanosoma cruzi -induced ROS is a potent inducer of SASP in NIH-3T3 fibroblasts. NIH-3T3 fibroblasts were infected overnight with culture-derived T. cruzi trypomastigotes (MOI 5:1). After washing step to eliminate extracellular parasites, cultures were treated with (A) 50% supernatants from non-infected or (B) T. cruzi -infected cultures (conditioned medium) plus 50% of fresh medium. After 3 days, the number of T. cruzi -infected or non-infected fibroblasts were quantified by optical microscopy. Dead cells were evaluated by Trypan blue exclusion assay. (C–E) NIH-3T3 fibroblasts were loaded with the probe DCFH-DA, washed and infected with T. cruzi concomitantly with addition of antioxidants NAC (20 mM) and DFO (40 µM) during infection period (MOI 5:1). After overnight infection, cultures were washed to eliminate extracellular parasites and cultured for additional 3 days. (C) Assessment of ROS accumulation after T. cruzi infection in NIH-3T3 fibroblasts by fluorescence. Results indicate arbitrary units of fluorescence. (D) Evaluation of IL-6 in culture supernatant by ELISA. (E) Evaluation of soluble SA-β-gal activity at pH 6.0 in cell lysates. (F) Assessment of extracellular parasitic load (trypomastigote forms) in culture supernatants over 10 days. Data are presented as the mean ± SE of at least six biological replicates and analyzed by unpaired, two-tailed Student’s t -test (C,D) , and non-parametric Mann–Whitney test (E) . For (A,B,F) , data were log-transformed and analyzed by Student’s t -test. * p
Article Snippet: In some experiments, the antioxidants NAC (20 mM; Sigma) or DFO (40 µM; Sigma) were diluted in sterile water and added or not to the cell cultures right after infection and washing step.
Techniques: Infection, Derivative Assay, Microscopy, Trypan Blue Exclusion Assay, Cell Culture, Fluorescence, Enzyme-linked Immunosorbent Assay, Activity Assay, Two Tailed Test, MANN-WHITNEY, Transformation Assay