Fluo 8 calcium flux assay kit

A Fluo-8 Calcium Flux Assay kit (Abcam) was used to measure intracellular calcium influx on a FLUOstar Omega microplate reader following the manufacturer’s protocols. Briefly, trypsinised HAP1 cells (3 × 10⁴) were seeded in each well of a 96-well plate. After 24 h, plates were washed twice in Ca²⁺-free HBSS supplemented with HEPES buffer (pH 7.2), and then the growth medium was replaced with 100 μl/well of the Fluo-8 dye solution. The plate was incubated at 37 °C for 30 min and then for another 30 min in the dark at room temperature. The loaded cells were then placed in the measurement position in the microplate reader. Changes in fluorescence from the Fluo-8 dye quantify changes in intracellular Ca²⁺ concentrations (excitation/emission 490/525 nm) after treatment with box jellyfish venom. Ca²⁺ influx was measured up to 45 min.

Intracellular Ca²⁺ levels were measured using the Fluo-8 Calcium Flux Assay Kit (Cat#ab112129, Abcam plc, CB2, 0AX, UK). T84 cells were seeded (1 x 10⁵ cells/well) and cultured for 24 h on 96-black well plates before incubation with Fluo-8 dye-loading solution at 37°C for 30 min and room temperature for 30 min. Cells were then incubated with DMSO (vehicle) or 20 μM of DHLV, followed by fluorescence intensity measurement (excitation wavelengths of 490 nm and emission wavelengths of 525 nm) using the multi-mode microplate reader (Biotek, Life Science, Inc., USA).

Intracellular Ca²⁺ in human OA chondrocytes or C28I2 cells was measured using Fluo-8 Calcium Flux Assay Kit (Abcam, ab112129) according to the manufacturer’s instructions. In brief, cells seeded in black walled 96-well plate (Corning, 3904) were loaded with Fluo-8 in Hanks’ Balanced Salt Solution for 30 min at 37 °C and 30 min at room temperature in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264. Ca²⁺ transients in chondrocytes were induced by 100 nM ATP in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264. Fluorescence was measured in a fluorescent plate reader with an excitation wavelength of 490 nm and emission wavelength of 525 nm, and expressed as the ratio of cytosolic fluorescence and initial intensity (F/F₀).

Cells were plated on a 96-well black plate pre-coated with poly-L-ornithine/laminin at a density of 15,000 cells/cm² and maintained for 7 days. Subsequently, the cells were incubated with a calcium indicator dye (Fluo-8 Calcium Flux Assay Kit, ab112129, Abcam, Cambridge, UK) for 1 h. After the incubation with the dye, the cells were kept in Hanks’ Balanced Salt Solution (HBSS) buffer. To assess the increase in intracellular calcium induced by caffeine, 10 mM of caffeine (Sigma-Aldrich, USA) in HBSS was added to the cells. In some experiments, 40 µM of cyclopiazonic acid (Sigma-Aldrich, USA) in HBSS was pre-incubated to deplete calcium in the endoplasmic reticulum . Intracellular calcium signaling was recorded at a frame rate of 2 Hz using a Zeiss LSM 900 confocal microscope (Carl Zeiss, Germany).

Lidocaine-derived organic compounds (EI137–EI641) were synthesized according to the reported methods at the Department of Pharmaceutical Organic and Medicinal Chemistry, Assiut University, Assiut, Egypt [13 (link)]. Their structures and purity were verified on the bases of spectral and elemental methods of analysis. CellTiter 96^® Aqueous One Solution Assay kit was purchased from Promega (Madison, WI, USA). Cytochrome C and PAF were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human IL-5 and IL-8 enzyme-linked immunosorbent assay (ELISA) kits were purchased from an R&D system (Minneapolis, MN, USA). Anti-CD16 antibody was purchased from Miltenyi Biotec (Sunnyvale, CA, USA). A human eosinophil cationic protein (ECP) ELISA kit was purchased from MyBioSource (San Diego, CA, USA). The P38 mitogen-activated protein kinase (MAPK) ELISA kit and Fluo-8 calcium flux assay kit were purchased from Abcam (Cambridge, UK). Human p-IκB-alpha and total IκB-α were purchased from RayBio (Norcross, GA, USA). Extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), and NF-κB InstantOne ELISA kits were purchased from Invitrogen (Carlsbad, CA, USA).

Calcium measurement was performed using the Fluo-8 calcium flux assay kit (Abcam, Cat# ab112129). Cells were seeded with an equal number of cells in 96-well plates and cultured until confluent, then treated with test chemicals for desired time. Subsequently, the supernatant was removed and replaced with 100 μL Fluo-8 dye-loading solution per well and incubated for 30 min at room temperature. Next, the fluorescence intensity was measured at Ex/Em of 490/525 nm using a fluorescence plate reader (Spectramax i3x, Molecular Devices, San Jose, CA, USA).

We seeded the HaCaT cells in 96-well microtiter flat bottom plate with 5 × 10⁴ cells/well and incubated for 24 h at 37 °C in 5% CO₂. The intracellular calcium measurement assays were performed using Fluo-8 calcium flux assay kit—no wash (Abcam) according to the protocol provided by the company. The HaCaT cells were treated with the neurochemicals (TRY, PEA, TYM, and DOP) and ICI, with and without EPI. We performed at least 3 times independent replications for each experiment.