Isolation and Flow Cytometric Analysis of Mesenteric Lymphoid Cells

Mesenteric lymphoid nods (MLN) were gently extruded through a 50 μm-mesh Nylon cell strainer (BD) to isolate mononuclear cells. Then, cells were suspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% of FBS, 2 mM l-glutamine, 50 U/mg penicillin, and 50 µ/mg streptomycin (Lonza, Levallois-Perret, France). For flow cytometry analysis, aliquots of 10⁶–10⁷ cells per sample were labelled with anti-CD3-FICT, anti-CD4-PerCP, anti-Tbet-APC and anti-Gata3-PE according to the manufacturer’s instruction (eBioscience). The cells were analyzed using flow cytometry (Accuri, BD) and CFlow Sampler software (BD Biosciences) as described previously^{30 (link)}.

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Martín R., Benítez-Cabello A., Kulakauskas S., Viana M.V., Chamignon C., Courtin P., Carbonne C., Chain F., Pham H.P., Derrien M., Bermúdez-Humarán L.G., Chapot-Chartier M.P., Smokvina T, & Langella P. (2023). Over-production of exopolysaccharide by Lacticaseibacillus rhamnosus CNCM I-3690 strain cutbacks its beneficial effect on the host. Scientific Reports, 13, 6114.

Publication 2023

Nylon cell strainer l-glutamine penicillin streptomycin anti-CD4-PerCP anti-Tbet-APC anti-Gata3-PE Accuri CFlow Sampler software

Anti cd3 Cells Eagle Flow cytometry Gata3 Glutamine Lymphoid Mesenteric Nods Nylon Penicillin Streptomycin

Corresponding Organization :

Other organizations : Microbiologie de l’alimentation au service de la santé, Universidade Federal de Minas Gerais, Danone (Netherlands), Nutricia Research (Netherlands)

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Variable analysis

independent variables

Isolation of mononuclear cells from mesenteric lymphoid nodes (MLN) using a 50 μm-mesh Nylon cell strainer

dependent variables

Phenotypic characterization of isolated mononuclear cells by flow cytometry, including expression of CD3, CD4, Tbet, and Gata3

control variables

Cell culture conditions: Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% of FBS, 2 mM L-glutamine, 50 U/mg penicillin, and 50 μ/mg streptomycin

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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