In-vivo cytotoxicity assay
RAW 264.7 macrophages were used as target cells for in-vivocytotoxicity assay. RAW 264.7 cell line was acquired from American Type
Culture Collection (ATCC TIB-71). Cells were cultured in supplemented
DMEM in an atmosphere of 5% CO2 and 95% humidity at 37
ºC. RAW 264.7 Macrophages were stimulated with 5 μg/mL of soluble
antigen of T. cruzi (H1 strain) for 2 h, consecutively were
stained with CFSE 2 μM (CFSE High) and were mixed in equal proportion
with non-stimulated macrophages previously stained with CFSE 0.2 μM
(CFSE Low). At day 49 p.i., a total of 4x106 mixed
macrophages (CFSE High and Low) were transferred intravenously to
non-infected, infected, TSA-1-C4+E6020-SE, Tc24-C4+E6020-SE and
TSA-1-C4+Tc24-C4+E6020-SE treated mice. Approximately 20 h later, when
mice were euthanized, a total of 10x106 spleen
mononuclear cells were recovered and resuspended in FACS buffer. Samples
were acquired using BD FACSVerse flow cytometer, and at least
1x106 events in macrophage cell gate were obtained
using FACSuiteTM software version 1.0.5. Data were analyzed in FlowJo
software version 10.0.7r2. Flow cytometry gating strategies for
cytotoxicity assay is presented in Supplementary Fig. 2 . The
percentage of cytotoxicity was determined using the following
formula40:
\begin{equation}
\left[\mathbf{1-}\frac{\mathbf{(\%CFSE\ }\text{High}\mathbf{\ INF\ /\ \%CFSE\ }\text{Low}\mathbf{\ INF)\ }}{\mathbf{(\%CFSE\ }\text{High}\mathbf{\ NI\ /\ \%CFSE\ }\text{Low}\mathbf{\ NI)}}\right]\mathbf{X\ 100\%}\nonumber \\
\end{equation}Percentage of cytotoxicity was calculated by the difference between CFSE
High versus CFSE Low in infected mice (INF) vs the differences of CFSE
High and Low in non-infected mice (NI).