Computational protocol: Alteration of Mevalonate Pathway in Rat Splenic Lymphocytes: Possible Role in Cytokines Secretion Regulated by L-Theanine

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Protocol publication

[…] According to the established method [], the lymphocytes cultured for 48 h were labeled with CFSE. In brief, the cells were pelleted and resuspended in prewarmed (37°C) PBS with 0.1% FBS. Freshly prepared CFSE was immediately added to the cell suspension at a final concentration of 5 μM and the cells were incubated for 10 min at 37°C. Excess CFSE was quenched by adding 10 volumes of ice-cold complete medium and incubating cells for 5 minutes on ice. After staining, the cells were washed twice in RPMI-1640 medium using centrifugation at 400 ×g for 5 min. This staining procedure was optimized so that even highly proliferated daughter cells were easily distinguishable from unlabeled cells during flow cytometric analysis. After the final wash, the cells were separated into five groups (Control, ConA, 0.25 mM L-theanine, 1 mM L-theanine, and 4 mM L-theanine treatments), resuspended with the complete medium, and treated with L-theanine solutions, subsequently transferred to a 96-well plate (Nunclon® Surface, Nunc, Roskilde, Denmark) using 100 μL/well, and then cultivated in a 5% CO2 incubator at 37°C for 120 h.The labeled cells were collected by centrifuging at 400 ×g at 4°C for 5 min and then used to measure percentage of the lymphocytes subsets which expressed CD3, CD4, and CD8 molecules by flow cytometry []. Cytometric monoclonal antibodies directed against surface antigens and presented on the lymphocytes containing CD3, CD4, and CD8 were immobilized. In brief, from each subject nine aliquots of 100 μL were prepared: five aliquots corresponding to five groups, respectively, were used for staining, three aliquots were used as control for the antibodies CD3+, CD4+, and CD8+, and one aliquot was used as negative control (no antibody incubated). The staining protocol was performed according to manufacturer's instruction of the antibodies, respectively, and the established method []. Aliquots were incubated for 30 min in darkness at 4°C, washed three times with 1 mL of FBS/PBS (400 ×g for 5 min at 4°C), and finally resuspended in 300 μL PBS and kept on ice until analysis.Cytometric analysis was performed on FACSAria™ II Cell Sorter flow cytometer (Becton, Dickinson Company, USA) in the Central Laboratory of Xiangya Hospital, Central South University, in accordance with the manufacturer's procedure. Acquisition was then performed on a FACSAria™ II Cell Sorter (Becton Dickinson China, China) with BD FACSDiva software (version 6.1.3). CD3+CD4+CD8+, CD3+CD4−CD8−, CD3+CD4+CD8−, CD3+CD4−CD8+, and total lymphocytes were identified by their classical forward scatter and side scatter signals and a minimum of 100,000 lymphocytes from each sample were collected in the gate. Data were analyzed with FlowJo software (version 8.3.2), and the results were finally expressed as percentage of positive cells (%). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Rattus norvegicus
Chemicals Amino Acids, Mevalonic Acid