Quantifying Intracellular Parasite Invasion

After treatment, infection and fixation, coverslips containing attached cells were washed with PBS, incubated for 20 minutes with PBS containing 2% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich) and processed for an inside/outside immunofluorescence invasion assay as described previously [27 (link)]. Briefly, cells were fixed and extracellular parasites were immune-stained using a 1:500 dilution of rabbit anti-T. cruzi polyclonal antibodies in PBS containing 2% (w/v) BSA (PBS/BSA) followed by labeling with Alexa Fluor-546 conjugated anti-rabbit IgG antibody (Invitrogen).
For evaluation of parasitophorous vacuole escape, after extracellular parasite staining, cells were permeabilized using a solution containing 2% (w/v) BSA and 0.5% (v/v) saponin (Sigma-Aldrich) in PBS (PBS/BSA/saponin) for 20 minutes. Host cell lysosomes were then immunostained using a 1:50 dilution of rat anti-mouse LAMP-1 hybridoma supernatant (1D4B; Developmental Studies Hybridoma Bank, USA) in PBS/BSA/saponin for 45 minutes followed by labeling with Alexa Fluor-488 conjugated anti-rat IgG antibody (Invitrogen), as described previously [30 (link)]. Subsequently, DNA from both host cells and parasites were stained for 1 min with 10 μM of DAPI (Sigma-Aldrich), mounted on glass slide and examined on an Olympus BX51, Zeiss, Apotome or Nikon Eclipse Ti.