MACSxpress Whole Blood Pan T Cell Isolation Kit, human

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	Pan-T cells were isolated from 30 mL whole blood using human MACSxpress Whole Blood Pan T Cell Isolation Kit (Miltenyi Biotec).

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Pan-T cells were isolated from 30 mL whole blood using human MACSxpress Whole Blood Pan T Cell Isolation Kit (Miltenyi Biotec).

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Pan-T cells were isolated from 30 mL whole blood using human MACSxpress Whole Blood Pan T Cell Isolation Kit (Miltenyi Biotec). Cells were extracellularly stained using anti-CD4 (AB_314079; RPA-T4), -CD8α (AB_10898322; RPA-T8), -CD45RA (AB_893358; HI100), -CD45RO (AB_2566542; UCHL1), -CCR7 (AB_10913812; G043H7), and -CD25 (AB_2561860; M-A251), all from BioLegend, followed by FACS sorting for naive (CCR7+CD45RA+CD45RO–) CD4+ T cells and CD8+ T cells, respectively, on an SH800. FACS-sorted naive CD4+/CD8+ T cells were stimulated with Dynabeads Human T-activator CD3/CD28 (Gibco, Thermo Fisher Scientific) at a 1:1 ratio on 96-well plates (75,000 cells/well) in 200 μL AIM V medium/well (Gibco, Thermo Fisher Scientific) for 5 days, in the last 24 hours in the presence of DMSO and 2 μM MS-275, respectively. For cytokine detection, activated cells were restimulated for 4 hours with PMA (25 ng/mL) and Iono (750 ng/mL), both from Sigma-Aldrich, in the presence of GolgiStop (BD Biosciences). For the treatment with SCFAs, FACS-sorted naive CD4+ T cells were stimulated with T Cell Activation MACSiBeads (Miltenyi Biotec) in presence of 20 ng/mL rhIL-12 for 5 days. Twenty-four hours after stimulation with beads, pentanoate was added to a final concentration of 3 mM. For cytokine detection, activated cells were restimulated for 4 hours with PMA and Iono in the presence of Brefeldin A (5 μg/mL, BioLegend). Subsequently, approximately 0.5 × 106 cells were surface stained using CD4 and CD8α; dead cells were excluded using Fixable Viability Dye eFluor 506 (Thermo Fisher Scientific) according to the manufacturer’s protocol. For intracellular cytokine stainings, cells were fixed with Cytofix Fixation Buffer (BD Biosciences), permeabilized with Perm/Wash Buffer (BD Biosciences), and stained according to the manufacturer’s protocol. For intracellular transcription factor stainings, cells were fixed and permeabilized using the Foxp3 Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cells were stained using the following antibodies: IFN-γ (AB_315230, AB_315236; 4S.B3) from BioLegend, RUNX3 (AB_2738969; R3-5G4) and granzyme B (AB_11154033; GB11) from BD Biosciences, and EOMES (AB_2574229; WD1928) from Thermo Fisher Scientific. Cells were measured with a BD LSRFortessa or BD LSRII cytometer and analyzed using FlowJo 10.2 software.

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