Phorbol myristate acetate (pma)

MAPK pathways were activated intracellularly via PKC with 1 μM to 1 nM PMA (Sigma–Aldrich; catalog no.: P1585). PMA treatments were applied as a 2° treatment in place of Bic. PMA was diluted in DMSO (Sigma; catalog no.: D2650) to achieve the variable PMA concentrations, so that the same DMSO load was added to each treated condition. PMA treatments were combined with 1 μM TTX (Calbiochem; catalog no.: 554412) to block membrane activity.

Peripheral blood samples were collected from control or ischemic stroke subjects with ischemic stroke within 3 days (acute stage) and 7–10 days (subacute stage) after stroke onset. Thereafter, mononuclear cells were isolated from the whole-blood specimens and immediately stained with fluorescent-labeled antibodies. In the case of IFN-γ staining, 2 × 10⁶ PBMC were cultured in the cell medium (RPMI 1640 + 10% heat-inactivated fetal bovine serum + 1% penicillin-streptomycin + 1 mM L-glutamine) for 4 h at 37°C in a CO₂ incubator in the presence of ionomycin (1 μM; Merck), phorbol-myristate-acetate (PMA, 10 ng/mL; Sigma), and GolgiPlug containing brefeldin A (1 μg/mL; BD biosciences) before flow cytometry analysis. For the staining of the intracellular molecules, cells were fixed and permeabilized using commercial kit (BD Pharmingen) according to the manual. All antibodies were ordered from Biolegend (San Diego, CA, USA), unless otherwise indicated. Antibodies used in this study included CD3 (UCHT1), CD56 (HCD56), CD69 (FN50), NKG2D (1D11), CD27 (O323), CD158 (HP-MA4), IFN-γ (4S.B3), and Perforin (dG9). Flow cytometry was performed using a FACS Aria III (BD Bioscience, San Jose, CA, USA), and data were analyzed by Flow Jo version V10 (flowjo.com).

After 10 days of incubation, each co-culture well was resuspended at 2 × 10⁶ cells per mL in 400 μL T cell medium and split into two aliquots (200 μL each). Cells were washed twice in FACS buffer and stained with the following antibodies (extracellular staining): FITC-CD8a (1:80)(Peprotech), Per55-I-A/I-E (1:200) (Peprotech), APC-TCRb (1:40) (Peprotech), at 4°C for 20 min. Cells were then washed twice in FACS buffer, fixed and stained (intracellular staining) for Granzyme B (PE-conjugated anti-GrB) (Peprotech), using the Cytofix/Cytoperm kit (BD), according to the manufacturer's instructions. PBMCs co-cultured with healthy organoids served as a negative control whereas PBMCs stimulated with 50 ng/mL PMA (Sigma-Aldrich) and 1 μg/mL Ionomycin (Sigma-Aldrich) served as a positive control; half of the contents of each co-culture well was incubated with PMA/Ionomycin solution for 5 h on ice.

THP1 cells were maintained in RPMI 1640 (Gibco, Grand Island, NY, USA) with a supplement of 10% FBS (Gibco) and 100 U/mL penicillin/streptomycin (Gibco). To induce monocytic differentiation, THP1 cells were plated at a density of 4 × 10⁵ /well on 6-well culture plate and pre-treated with PMA (Sigma) for 48 h. After PMA treatment, cells were stabilized for another 24 h in the maintenance media. Macrophages were then polarized in M1 macrophages by incubation with 20 ng/mL of IFN-γ (Peprotech) and 10 μg/mL of LPS (Sigma) for 5 days. PMA-pretreated cells were cultured within the maintenance media for 5 days without any lineage-specific stimulants for the spontaneous polarization towards M2 macrophages. To assess the Ech A impact on macrophage polarization, Ech A or vehicle was also treated during the culture period. Five days later, the culture supernatant was collected and secreted cytokine levels were estimated using human TNF-α and the IL-10 Duoset ELISA kit (R&D system, Abingdon, UK).

For detection of intracellular cytokines, cells after coculture were activated with PMA (50 ng/ml; Sigma) and ionomycin (1 μM;Merk) for 6 h in the presence of GolgiPlug (1 μl/ml BD Biosciences) for the final 4 h. Cytokines in coculture supernatants were analyzed by LEGENDplex Th cytokine panel (BioLegend).
For activation of eTAC, cDC and pDC, purified subsets were cultured at 5 × 103 cells per well in the presence of LPS (100 ng/ml; Sigma), poly(I:C) (25 μg/ml;Sigma) and R848 (25 μg/ml; Invivogen), or with PMA (50 ng/ml; Sigma) and ionomycin (1 μM;Merk). Culture supernatants were harvested after 24 h and analyzed by a custom LEGENDPlex human inflammation panel (BioLegend).