Diseño de un panel multicolor para evaluar moléculas intracelulares y de superficie mediante citometría de flujo

Abstract

* Los autores contribuyeron de igual manera en el presente estudio.Institución donde se llevó a cabo el trabajo: Pontificia Universidad Javeriana, Bogotá, Colombia.Introducción. La citometría de flujo permite detectar la presencia de moléculas intracelulares y de superficie, de forma simultánea sobre cada célula. Objetivo. Describir un método para la construcción armónica de un panel multicolor con 11 parámetros para el análisis fenotípico y funcional de linfocitos T (LT) CD8 + por citometría de flujo. Materiales y métodos. Para la construcción del panel multicolor, se seleccionaron las moléculas y se titularon los conjugados con fluorocromos para la determinación de CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 y CD127, en células mononucleares de sangre periférica. Para la evaluación del panel, se hizo la construcción progresiva adicionando uno a uno los conjugados y la fluorescencia menos uno (FMO). Este método fue aplicado para células ex vivo y para evaluar la producción de IFNγ, IL-2 y TNFα frente al estímulo con la enterotoxina B de Staphylococcus aureus (SEB) y al antígeno crudo de Trypanosoma cruzi. Finalmente, se procedió al análisis de las subpoblaciones de LT CD8 + ex vivo en individuos sanos. Resultados. La evaluación de las moléculas con los conjugados no mostró interferencia en las señales de fluorescencia. Las frecuencias de las subpoblaciones de LT CD8 + evaluadas fueron cercanas a los valores reportados en otros estudios. Además, se observó que la frecuencia de LT CD8 + productores de IFNγ, IL-2 y TNFα fue mayor a las seis horas de cultivo con SEB y con el antígeno crudo de T. cruzi. Conclusiones. El método aplicado para la construcción del panel multicolor permite obtener frecuencias de las subpoblaciones de LT CD8 + que corresponden a lo reportado en la literatura científica. Objective: To describe a method for building up a harmonic multicolor panel with 11 flow cytometry parameters for phenotypic and functional analysis on CD8 + T lymphocytes. Materials and methods: For the multicolor panel construction, we selected the molecules and titred conjugated antibodies with fluorochromes for CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 and CD127 determination in peripheral blood mononuclear cells (PBMC). To evaluate the panel, the conjugated antibodies were gradually added one by one and fluorescence minus one (FMO) test was performed. This method was applied to assess ex vivo subpopulations of T cells and the production of intracellular IFNγ, IL-2 and TNFα using polyclonal stimulation with enterotoxin B from Staphylococcus aureus (SEB) and antigen-specific cells with crude Trypanosoma cruzi antigen. Finally, the ex vivo CD8 + T lymphocyte subpopulations frequencies were analyzed in healthy individuals. Results: The evaluation of the selected molecules and conjugates did not show interference in the fluorescence signals and detection. The frequencies of CD8 + T cells evaluated were similar to the values reported in other studies. Additionally, we observed that the frequency of CD8 + T lymphocytes

Low Frequency of Circulating CD8+ T Stem Cell Memory Cells in Chronic Chagasic Patients with Severe Forms of the Disease

“…All conjugated antibodies were titrated, and each multicolour panel of conjugates was evaluated as previously described [14] . To evaluate the frequency of CD8 + T cell subsets, one million PBMCs were stained with the viability marker for 20 min in the dark at room temperature and then washed with PBS 0.001 M pH 7.4 (1X PBS) (Eurobio; Les Ulis, France).…”

“…To evaluate the cytokine production of CD8 + T cell subsets, one million PBMCs were cultured with anti-CD28 and anti-CD49d antibodies and incubated for 6 hours in the presence of brefeldin A (BD Biosciences, San Jose, CA, USA) with Staphylococcal enterotoxin B (SEB) (Sigma-Aldrich; Saint Louis, MO, USA), Trypanosoma cruzi trypomastigote lysate or medium. Parasite lysate was obtained as previously described [14] . First, cells were stained with the viability marker and then with surface antibodies against CD3, CD8, CD45RA, CCR7 and CD95 molecules for 30 min in the dark at 4°C.…”

“…Analysis was performed using FlowJo 9.3.2 (Tree Star; Ashland, OR, USA), Pestle 1.7 (National Institutes of Health (NIH), Bethesda, MD, USA) and SPICE 5.3 (NIH) software [15] . Dead and doublet cells were excluded from the analysis, as previously described [14] . A positive cytokine response was defined by subtracting the cytokine background (cells cultured with medium) from a frequency of >0.05% for each CD8 + T cell subset (average frequency of the response of CD8 + T cells from HDs cultured with parasite lysate plus 3 SD).…”

Impact of benznidazole treatment on the functional response of Trypanosoma cruzi antigen-specific CD4+CD8+ T cells in chronic Chagas disease patients

“…Conjugated Abs for intracellular staining included the following: cytotoxic T lymphocyte antigen-4 (CTLA-4)-PE-Cy5 (clone BNI3), granzyme B-PE-CF594 (clone GB11), interferon-gamma (IFN-γ)-PE-Cy7 (clone B27), interleukin-2 (IL-2)-APC (clone MQ1-17H12), tumor necrosis factor-alpha (TNF-α)-Alexa Fluor 488 (clone MAb11) (BD Biosciences, San Diego, CA), and perforin-PE (clone B-D48) (Abcam, Cambridge, UK). All conjugated Abs were titrated and multicolor panels for flow cytometry assays were approached as previously reported [ 38 ]. The following purified (No azide/Low endotoxin) Abs were used in cultured cells: CD28 (clone CD28.2) and CD49d (clone 9F10) (BD Biosciences).…”

Expression of inhibitory receptors and polyfunctional responses of T cells are linked to the risk of congenital transmission of T. cruzi

“…A LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen Molecular Probes, Eugene, OR) was used for dead cell exclusion. All conjugated Abs were titrated as previously reported [22]. …”