Estudo comparativo de métodos de isolamento e estimulação ex vivo de pbmc de equinos para análise por citometria de fluxo

Abstract

Magalhães, V. L. P. Comparative study of horse PBMC isolation and ex vivo stimulation techniques for flow cytometry analysis. 2021. XP. Dissertação (Mestrado em Ciência Animal nos Trópicos) – Escola de Medicina Veterinária e Zootecnia – Universidade Federal da Bahia, 2021. The isolation and stimulation of peripheral blood mononuclear cells (PBMC) with mitogens is a technique commonly used in scientific research. Recent studies have shown that variations in horse PBMC isolation protocols can interfere in several cell parameters, such as yield, proliferation capacity and cell population composition, which may lead to underestimation of obtained data. In this study, we aimed to standardize a modified protocol for isolation of PBMC from horses, as well as lymphoproliferation after ex vivo stimulation with different concentrations of concanavalin A (Con A), analyze it by flow cytometry and compare it to a conventional protocol. Blood from 12 adult horses was used. The conventional protocol (Protocol 1) consisted of isolating PBMC from whole blood, and the modified protocol (Protocol 2) consisted of obtaining PBMC from blood plasma collected after erythrocyte sedimentation. In both protocols, PBMC separation was performed using a Ficoll-Paque Plus density gradient. Following the Protocol 2, immunophenotyping of CD4 and CD8 was performed to verify if the population of cells obtained consisted of lymphocytes. After isolation and cell yield analysis, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with Con A at concentrations of 1.25, 2.5 and 5.0 µg/mL. Cells were acquired in a flow cytometer and the cell viability and lymphoproliferation results were analyzed using the FlowJo program. The pilot assays results suggested that yield, cell viability and lymphoproliferation were higher in Protocol 2 compared to Protocol 1 and that the best Con A concentration was 2.5 µg/mL. In the final assay, cell viability was similar between the two PBMC separation protocols. Cell yield (1.4x107 cells/mL ± 0.5; P=0.0159) and lymphoproliferation rate (40.78% ± 12.4; P=0.0278) were higher in Protocol 2 compared to the conventional protocol. The data obtained show that the isolation of PBMC from horse plasma was superior to the conventional protocol, and the method used can influence the cellular parameters under stimulation with mitogen.