Raw data from data presented in Figures 1, 3, 4 and 5.

Published: 18 November 2024| Version 1 | DOI: 10.17632/cm93t45psx.1
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Description

The data shown in this repository refer to those presented in Figure 1.

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- POMVs and SEVs extractions (Figure 1). Briefly, overnight cultures of Pseudomonas aeruginosa and Staphylococcus aureus were seeded in fresh LB and grown to the late stationary phase (20 h). The bacterial cultures were centrifuged and the BMVs-containing supernatants were filtered, followed by concentration through ultrafiltration in a dialysis membrane. For OMV isolation, the filtered supernatants were ultracentrifugated. The vesicles were diluted in a 10mM HEPES/0.85% NaCl buffer and stored at -80 ºC. - Nanoparticle Tracking Analysis (NTA) of the membrane vesicles (Figure 1): Size-distribution analysis and quantification of BMV preparations were performed on a NanoSight. Both scatter and capture settings such as focus, camera, and gain settings were optimized to make particle tracks visible. Then measurements were obtained in triplicate and analyzed using NanoSight software. - Bradford assay of the POMVs and SEVs (Figure 1): The total protein content of POMVs and SEVs was determined with bovine serum albumin (BSA) as a reference for the calibration curve. - Leishmania amazonensis-infected RAW 264.7 macrophages treated with POMVs or SEVs (Figures 3 and 4): macrophages were seeded in a 24-well plate and were incubated overnight at 37 °C, 5% CO2. The macrophage co-cultures were then incubated for 24 h with the parasite. The BMVs were then inoculated in the wells. The coverslips in the plate wells were fixed and stained with Giemsa. Adherent macrophages with a structurally preserved nucleus and cytoplasm were considered viable and were quantified in 40 representative fields to assess viability, the number of L. amazonensis-infected macrophages, and the number of intracellular amastigotes. The percentage of infected macrophages was calculated as the ratio of the total macrophages counted, and the number of amastigotes per infected macrophage was calculated by dividing the total number of intracellular amastigotes by the total number of infected macrophages. The infection index was calculated as the product of the percentage of infected macrophages and the number of amastigotes per infected macrophage. - Cytometric Bead Array (CBA) (Figure 5): IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α were quantified by flow cytometry using BD cytokine kit, according to the manufacturer’s instructions. - Enzyme-Linked Immunosorbent Assay (ELISA) (Figure 5): IL-1β were quantified using a Rat IL-1β/IL-1F2 DuoSet ELISA Kit (R&D Systems) and all procedures followed the manufacturer's instructions. - Statistical analysis: For the statistical analysis, media ± SD was calculated, and variance analysis was performed between experimental situations, on the software GraphPad Prism® Ver. 9.0.2. The statistical significance was verified through the t-Student test with Welch’s correction, when applicable, and considered acceptable when p≤ 0,05.

Institutions

Universidade Estadual de Campinas Instituto de Biologia

Categories

Microbiology, Bacterial Protein, Extracellular Vesicle

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