Computational protocol: Dopaminergic Stimulation of Myeloid Antigen-Presenting Cells Attenuates Signal Transducer and Activator of Transcription 3-Activation Favouring the Development of Experimental Autoimmune Encephalomyelitis

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Protocol publication

[…] To analyse cytokine production, cells were restimulated with 1 mg/ml ionomycin and 50 ng/ml PMA for 4 h in the presence of 5 mg/ml brefeldin A. For intracellular staining, cells were first stained with a Zombie Aqua fixable viability kit (BioLegend), followed by staining for cell surface markers. Intracellular staining was done with the Foxp3 staining buffer set (eBioscience). Data were collected with a FACSCanto (BD Biosciences) and analysed with FlowJo software (Tree Star). [...] Analysis of DRs on DCs and monocytes was performed according to a previously established method () with modifications. Briefly, 200 μl aliquots of whole blood were prepared and erythrocytes were removed by means of a lysis buffer [composition (g/l in ultrapure water): NH4Cl 8.248, KHCO3 1.0, EDTA 0.0368]. Incubation was performed at RT for 5 min, during which samples were gently vortexed. Samples were then centrifuged at 600 g for 5 min at RT, supernatants were removed, and cells were washed one time in 1 ml of PBS [composition (g/l in ultrapure water): NaCl 8.4, Na2HPO4 1.424, NaH2PO4 0.276] at pH 7.4 supplemented with 1% BSA (Sigma, Italy) (PBS/BSA), once more centrifuged at 600 g for 5 min at RT, and finally resuspended in 100 µl PBS/BSA. From each subject, 13 aliquots of whole blood were prepared and processed for evaluation of DCs (6 aliquots: 5 for DRs staining, 1 as control for the secondary Ab) and of monocytes (7 aliquots: 5 for DRs staining, 1 as control for the secondary Ab, and 1 for the anti-CD16 Ab isotype control).The staining protocol consisted of two steps. During the first step, each aliquot was treated with 5 µl Fc block solution for 10 min at RT to prevent any unwanted binding of anti-human Ab to Fc receptors and thereafter stained for one of the five DRs by an indirect labelling procedure (primary Ab + secondary Ab). Aliquots were incubated with the primary anti-DR Ab for 30 min on ice in the dark, washed one time with PBS/BSA at 600 g for 5 min at RT, and resuspended in 100 µl PBS/BSA containing the secondary Ab and incubated for 30 min on ice in the dark. Aliquots were then washed (600 g for 5 min at RT) and once more resuspended in 100 µl PBS/BSA. During the second step, the aliquots for evaluation of DCs were incubated with a cocktail of anti-human Lin1, human leucocyte antigen DR (HLA-DR), CD123, and CD11c, for the identification of total circulating DCs (Lin1-/HLA-DR+), plasmacytoid DCs (pDCs, CD123high/CD11c−), and myeloid DC (mDC, CD123low/CD11chigh), whilst the aliquots for evaluation of monocytes were incubated with a cocktail of anti-human CD45, HLA-DR, CD14, and CD16 for the identification of classical (CD14high/CD16−), non-classical (CD14low/CD16high), and intermediate (CD14high/CD16+) monocytes. As a control of isotype, cells were stained with an IgG1k of irrelevant specificity instead of the anti-human CD16 Ab. All aliquots were incubated for 20 min in dark at RT, washed with 1 ml of PBS/BSA (600 g for 5 min at RT), finally resuspended in 600 µl PBS and kept on ice until analysis. The complete list of Ab used in the protocols together with their working dilutions is shown in Table S1 in Supplementary Material.Acquisition and analysis were performed on a BD FACSCanto II flow cytometer (Becton Dickinson, Milan, Italy) with BD FACSDiva software (version 6.1.3). The gating strategies used to identify DCs and monocytes are shown in Figures S9 and S10 in Supplementary Material, respectively. The results were finally expressed as absolute numbers (103/mm3) as well as percentage of positive cells (%), as well as mean fluorescence intensity (MFI) of positive cells, calculated as the difference between MFI in anti-human DR Ab stained aliquots and aliquots stained with the secondary Ab alone. […]

Pipeline specifications

Software tools FlowJo, BD FACSDiva
Application Flow cytometry