Phorbol myristate acetate (pma)

DPSCs were obtained from a patient with CS and healthy donors with approval by the Committee of Ethics, Nippon Dental University School of Life Dentistry, Tokyo. Informed consent was obtained from the patient. The dental pulp tissue was enzymatically digested as described in a previous study (Matsui et al., 2018). The cells were cultured in serum‐based minimum essential medium alpha (MEMα; Gibco/Thermo Fisher Scientific) supplemented with 20% fetal bovine serum (FBS; SAFC Biosciences; Gibco/Thermo Fisher Scientific), 100 µM l‐ascorbic acid phosphate magnesium salt n‐hydrate (Wako Pure Chemical Industries), 2 mM l‐glutamine (Gibco/Thermo Fisher Scientific), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco/Thermo Fisher Scientific) at 37°C with 5% CO₂ until Passage 1. Subsequently, the cells were cultured in STEMPRO® MSC SFM (Gibco/Thermo Fisher Scientific), a serum‐free medium for mesenchymal stem cell culture. The medium was changed every 2 days. At confluency, they were subcultured at a split ratio of 1:2 by gentle separation with TrypLE™ Express solution (Gibco/Thermo Fisher Scientific) at room temperature. To analyze the response to PMA, the cells were cultured in 60‐mm dishes (Corning), treated with 2.5 nM PMA (LC Laboratories), and harvested in centrifuge tubes. Cells cultured without PMA served as controls.

To explore the metabolic pathway leading to reorganization of intracellular Them1, various treatments were applied for 4 h to cultured iBAs as follows: Norepinephrine (1 μM; Sigma Aldrich; St. Louis, MO), a neurotransmitter, was used to mimic cold exposure. Forskolin (1 μM; Selleckchem, Houston, TX), a membrane permeable labdane diterpene produced from the Coleus plant, was used to activate adenylyl cyclase. This treatment, through the activation of cAMP, activates PKA. PMA (3 μM; LC Laboratories, Woburn, MA) was used to activate PKC. NE, adenylyl cyclase, and PKC were selected for activation as they represent three key control points in the pathway allowing the characterization of all upstream regulators. Pathway inhibitors were added 1 h prior to activation and were continued throughout the 4 h activation period as follows: PKI [14–22] myristoylated (0.5 μM; Invitrogen, Camarillo, CA), a synthetic peptide inhibitor of PKA, was added to inhibit PKA activation. Atglistatin (40 μM; Selleckchem, Houston, TX) was used to selectively inhibit the processing of triacylglycerol to fatty acids via the formation of diacylglycerol by ATGL. Ruboxistaurin, or LY333531, (2 μM; Selleckchem, Houston, TX), an isozyme-selective inhibitor of PKC that competitively and reversibly inhibits PKC-βI and PKC-βII, was added to inhibit PKCβ activity.

The PKC peptide inhibitor sc-3088 with the sequence FARKGALRQKNV (final concentration: 1 μM; Santa Cruz Biotechnology, Dallas, TX, USA) and the PKC activator PMA (final concentration: 80 nM; LC Laboratories, Woburn, MA, USA), both dissolved in dimethyl sulfoxide (DMSO), were added to the cell culture medium. DMSO served as a negative control.

Tumors were cut into small pieces, digested for 30 min at 37°C using Collagenase type IV (600U) (Sigma) and (400U) DNAse (Sigma). Next, tissue fragments were dissociated using a gentleMACS Dissociator, and filtered through a 100 μm cell strainer, washed with PBS containing 2 mM EDTA and 1% FCS, centrifuged and stained. Spleens were forced through the cell strainer (70 μm) and cells were centrifuged (500g) at 4ºC. When necessary, erythrocytes were lysed using a buffer containing 155 mM NH₄Cl, 10 mM NaH₂CO_3, and 0.1 mM EDTA, pH 7.3. For staining, cells were blocked in 5% normal rat serum and stained with fluorescently labeled monoclonal antibodies: anti-CD11b (M1/70, 53-0112-82), anti-Ly6C (HK1.4, 17-5932-80), anti-CD200R (OX110, 12-5201-82), anti-MHC-II (M5/114.15.2, 25-5321-80) (eBioscicence, USA), anti-F4/80 (BM8, 123118) (BioLegend), anti-TNF-α (XT22, 554419), anti-IFN-γ (XMG1.2, 563376) (BD Pharmingen). For intracellular staining of TNF-α or IFN-γ, cells were first stimulated with PMA (LC Laboratories) / ionomycin (Thermo Fisher), and GolgiStop (BD Pharmingen) for at least 5 hrs and subsequently stained using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.

Capture and detection antibodies from the feline IFNγ Development Module (R&D Systems) were used with MultiScreen-IP 96-well plates (MAIPSWU10; MilliporeSigma) to quantify IFNγ-producing mucosal lymphocytes after stimulation with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL; LC Laboratories, Woburn, MA) and ionomycin (300 ng/mL; LC Laboratories) or with FIPV antigen (60 µg/mL; IVD Technologies, Santa Ana, CA, USA) as previously described [16 (link)]. The protocol was modified with the use of 2.5 × 10⁴ cells/well (PMA/ionomycin) or 2 × 10⁵ cells/well (FIPV antigen) incubated for 40 h at 37 °C, 5% CO₂. Plates were scanned with an ImmunoSpot S5 Analyzer, and analysis was performed by CTL.