Phorbol myristate acetate (pma)

BMDNs were purified as previously described (Swamydas et al. 2015 ). In brief, bone marrow cells were resuspended in 1mL of ice-cold PBS and layered on top of a pre-layered solution of Histopaque 1119 (3mL, 11191, Sigma) on the bottom and Histopaque 1077 (3mL, 10771, Sigma) on top. Cells were centrifuged at 500xg for 30min at room temperature without deceleration. BMDNs were collected at the interface of the Histopaque layers, and purity was assessed by flow cytometry. For PMA activation, BMDNs were treated with PMA (100nM, Abcam) for 15min, washed, and cultured for conditioned media collection.

Human peripheral blood-derived monocytes THP-1 (ATCC^R TIB-202™, VA, USA) was added to pre-warmed ATCC-formulated RPMI-1640 medium, supplemented with 10% FBS and 0.05 mM 2-mercaptoethanol to reach the desired cell density. After reaching a cell density of ~1 × 10⁶ cells per mL, the suspension media was collected and centrifuged. The cell pellet was resuspended into an osteoclast differentiation media constituting of RPMI 1640 supplemented with 40 ngml⁻¹ phorbol 12-myristate 13-acetate (PMA, Abcam, Cambridge, UK), 10 ngml⁻¹ receptor activator of nuclear factor-kappa β ligand (RANKL, Abcam), 10 % FBS, antibiotic/antimycotic, and non-essential amino acids (Gibco, USA) (16 (link)–18 ), mixed with hMSC cells in different ratios of 2:1 and 3:2 hMSCs:THP1 (referred as 2:1 hMSCs:THP1 and 3:2 hMSCs:THP1) and pipetted into each well of a U-bottom 96-well plate to facilitate formation of heterocellular spheroids (~400 μm in diameter) with ~15,000 cells per well. Cultures were maintained in a mixed media of osteoblast and osteoclast differentiation media at the same ratio as of the cells in heterocellular spheroids at 37 °C under humidified atmosphere of 5% CO₂. Cell medium was changed every 2–3 days during the entire course of the experiment.

NMDA (CAT No: HB0454) and XE-991 (M channel/Kv7 blocker, CAT No: HB1090) were purchased from Hellobio (Princeton, NJ, USA) and were dissolved in water to make a stock solution. They were delivered into the recording chamber at their final concentrations through a syringe pump. PKC activator, PMA (CAT No: ab120297) and PKC inhibitor, GF 109203X (Bisindolylmaleimide 1 (Bis-1), CAT No: ab120844) were purchased from Abcam (Cambridge, UK) and were dissolved in DMSO to make a stock solution. They were added to the incubation solution at a concentration of 1.0 μM for PMA and 2.5 μM for Bis-1. The following agents were dissolved in DMSO to make a stock solution and were added to the internal solution. CaM antagonist, W-7 (CAT No: ab143768) was obtained from Abcam, and PIP2 (CAT No: P-4508-2) was purchased from Echelon Bioscience (Salt Lake City, UT, USA). LY294002 (CAT No: HY-10108, PI3K inhibitor) and AS605240 (CAT No: HY-10109, a specific inhibitor of the PI3Kγ) were purchased from MCE (Monmouth Junction, NJ, USA). Go6976 (CAT No: ab141413, a specific inhibitor of PKCα and PKCβ) and LY333531 (CAT No: ab219866, selective PKCβ inhibitor) were purchased from Abcam.