Ionomycin

To increase intracellular [Ca²⁺], cells were incubated in 140 mM NaCl, 5 mM KCl, 25 mM HEPES pH 7.4, 10 mM glucose, 1–5 mM CaCl₂, and 3 μM ionomycin (Cayman Chemicals) for 5 min at 37°C.
Cross-linking of proteins in HEK293 cells was carried out in 2 ml PBS (GE Lifesciences) with 0.3 mM DSS (Thermo) for 20 min at room temperature. After the reaction was quenched with 50 mM Tris for 15 min, the cells were lysed in 150 mM NaCl, 20 mM HEPES, 0.5% Triton X-100, and EDTA-free protease inhibitors (Roche). Lysates were cleared by centrifugation and subsequently analyzed by Western blotting.
For cross-linking of his₆PICK1, 100 nM purified protein was diluted in 150 mM NaCl, 20 mM HEPES pH 7.4, 5 mM HEDTA, and CaCl₂ at a concentration calculated using the MaxChelator online tool to give required [Ca²⁺]_free. [Ca²⁺]_total were 0.75 mM, 1.5 mM, 2.5 mM, and 3.4 mM to give [Ca²⁺]_free of 2, 5, 12, and 26 μM, respectively. 10 μM DSS was added for 20 min at room temperature before the reaction was quenched with 50 mM Tris for 15 min. The samples were subsequently analyzed by Western blotting.

AgNPs with a diameter of 20 nm were procured from NanoComposix (San Diego, CA). For most studies, we used a concentration of 25 μg/ml based on our previous findings and others [12 (link), 21 (link)]. AgNPs are suspended in citrate at a concentration of 1 mg/ml. Anti p-Tyr, p-Ser/Thr, p/t-plcγ1, p/t-PI3K were purchased from Cell Signaling (Beverly, MA), antibodies against SR-BI/II were purchased from LS BioSciences (Seattle, WA) and Thermoscientific (Waltham, MA). Cy^TM5 Annexin V (BD Pharmingen) was purchased from BD Biosciences (San Jose, CA) and propidium iodide was purchased from Invitrogen (San Diego, CA). Inhibitors and reagents used in our studies include SR-B1 inhibitor 2-(2-butoxyethyl)-1-cyclopentanone thosemic-arbazone (Blt2) (Chembridge Corp., San Diego, CA, USA), Synta (compound 66) (Aobious INC, Gloucester, MA), N,N-Dimethylsphingosine (DMS), Wortmannin, U-73122, Ionomycin (Cayman Chem, Ann Arbor, MI), Ro 31–8220 (Abcam, Cambridge, MA). Concentrations of inhibitors were based on previous literature [22 (link)–26 (link)].

A total of 10⁶ PBMC or remixed PBMC fraction (containing 1×10⁵ recombined dye-labeled NK cells prepared as described under cell tracing method) was co-cultured with K562 or U937 target cells (obtained from ATCC) in 500 μl cell culture medium for 6 hours at 37°C using an optimized effector:target ratio of 5:1. For CD107a expression, the CD107a mAb was added to the cells at the beginning of the culture. In order to block degradation of reinternalized CD107a and cytokine secretion, respectively, 2 μ molar monensin and 10 μg/ml of brefeldin A (GolgiStop and GolgiPlug, BD Biosciences) were added to the culture 1 h after start of the co-culture. In certain cases 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Merck, Darmstadt, Germany) and 1 μg/ml ionomycin (Cayman Chemical Co., Ann Harbor, MI) were added in addition to achieve a full activation. After incubation for additional 5 h, cells were then first stained on the surface with anti-CD56 and -CD3 antibodies. Then fixation and permeabilization of the cells were performed using Cytofix/Cytoperm solution (BD Biosciences). To assess cytokine production, the fixed and permeabilized cells were further stained with antibodies for IFN-γ, TNF-α, IL-10 and IL13 (Suppl. Table 2). Then flow cytometry was performed. As controls, PBMC were cultured without target cells to display their spontaneous CD107a expression and cytokine production.

Medical Borneol was obtained from Shanghai Hongqiao Traditional Chinese Medicine Co., Ltd. (+)‐Borneol (simply called Borneol in this study), (±)‐menthol (simply called menthol in this study), complete Freund's adjuvant, and capsaicin were purchased from Sigma‐Aldrich. Formalin was purchased from Xilong Chemical Co. AMTB, naloxone, and LY341495 were obtained from TOCRIS. Bicuculline was obtained from Tokyo Chemical Industry. Muscimol was obtained from Enzo Life Sciences. ATP disodium salt was obtained from Sangon Biotech. Ionomycin was purchased from Cayman Chemical. Morphine hydrochloride was obtained from Northeast Pharm.

For intracellular staining, T cells were restimulated with 5 ng/ml PMA (Cayman Chemical) and 200 ng/ml ionomycin (Cayman Chemical) for 4 h in the presence of GolgiPlug (BD Pharmingen). Subsequently, surface staining was performed at 4°C for 30 min. Cells were fixed and permeabilized using Cytofix/Cytoperm plus Fixation/Permeabilization Kit (BD Pharmingen). For analysis of intranuclear markers, cells were fixed and stained using the FoxP3 intranuclear staining kit (eBioscience), again incubating cells at 4°C for 30 min both during fixation and intranuclear staining. Antibodies are summarized in Table 1. Analysis was performed using a Gallios Flow Cytometer (Beckman Coulter) and results were analyzed with FlowJo software (Tree Star).