Calcein am

Opening of the mPTP was monitored by loading cells with calcein‐AM (Sigma‐Aldrich) and CoCl₂.²³ H9c2 cells were first incubated with calcein‐AM (2 μmol/L) and CoCl₂ (1 mmol/L) for 35 minutes at room temperature and then washed with CoCl₂. Basic fluorescence was measured at ex/em wavelengths of 488/515 nm. Subsequently, cells were treated with H₂O₂ (100 μmol/L) for 120 minutes at 37°C and fluorescence was measured at different time‐points. Opening of the mPTP was indicated by the abrupt loss of fluorescence and expressed as a percentage of the baseline fluorescence intensity.

Cell viability staining was performed by calcein-AM / PI double staining. A cell density of 1 × 10⁵ cells (MCs, MG) were seeded on round cover slips (12 mm diameter) and were treated with 2,5 μg/ml 5A1 for 24 h, 48 h, 72 h and 96 h, respectively. For calcein-AM / PI double staining, cells were incubated in 1 μM calcein-AM (Sigma-Aldrich, Seelze, Germany) plus 1 μM PI (Sigma-Aldrich, Seelze, Germany) for 30 min at 37°C in the dark. Subsequently, cells were fixed with 4% paraformaldehyde for 15 min before placing cover slips upside down on an object slide using mounting medium (Dako, Hamburg, Germany). Fluorescence signals were analysed with Leica DM500B (Leica, Wetzlar, Germany).

To determine the localization and spread of the cells on the bone slices, a detection with calcein-AM (Sigma-Aldrich, Munich, Germany) was made before detaching of the cells. calcein-AM (3 µM, Sigma-Aldrich) was added to the medium and incubated for 30 min at 37 °C, 5% CO₂. During this period, the calcein-AM is transported through the cell membrane into the cell where it is converted into calcein by esterase activity of the living cells. This forms a complex with calcium ions in the cytoplasm, resulting in optically visible green fluorescence.
Images were thus taken by fluorescent microscopy and heavily overgrown areas were marked for examination with the scanning electron microscope (Thermo Scientific, Waltham, MA, USA).

CD20-positive cells were harvested, suspended in complete medium to yield 10⁶ cells/ml and calcein-AM (Sigma) was added to the final concentration of 1 µg/ml. After 30 min incubation at standard culture conditions, cells were washed with PBS buffer with Ca²⁺/Mg²⁺ (Biowest), loaded into the V-shape wells of 96-well microplate (Nunc) at 10⁵ cells (or more, as indicated separately in the text) per well and pelleted. Pellets were overlaid with PBS w. Ca²⁺/Mg²⁺ containing desired concentration of ofatumumab (GlaxoSmithKline) and NHS, in a total volume of 50 µl. Microplates were incubated for 30 min. at 37 °C and shaken at 650 rpm, then overlaid with another 50 µl of PBS buffer and centrifuged. Eighty microliter of the supernatant was transferred into flat-bottom plate and fluorescence 485/515 nm was measured in Synergy H1 (Biotek) reader. Fluorescence readout obtained for cells loaded with calcein-AM and lysed with 2% NP40 (Sigma) was considered as full lysis.

Apoptosis was estimated in both cell lines by using bioluminescent test based on caspase 3/7 activity measurement. Cells from both analyzed lines were seeded in 96-well white plates and cultured for 24 h. After that time, cells were subjected to presented range of concentrations of LL for 24 and 48 h. After a specified incubation time, medium was removed and Caspase-Glo^® 3/7 assay was conducted according to the manufacturer’s instructions. The assay is based on the measurements of the luminescence level, which is proportional to the activity of caspases in analyzed cell lines. GloMax^®-Multi Microplate Multimode Reader was used in order to measure luminescent signal in samples. The study was performed in triplicate taken to ensure consistent results were obtained.
The average level of apoptosis was also estimated by using fluorescent microscopy method with the application of propidium iodide and calcein-AM dyes (Cell stain double staining kit containing propidium iodide and calcein—AM, Sigma-Aldrich, St. Louis, MO, USA).

To monitor the diffusion of fluorescent molecules through gap junctions (dye coupling), we used a dye transfer assay. For this experiment, NMCs treated or not with 100 nM Apelin-13 for 48 h and loaded with the gap junction permeant dye, Calcein-AM (FITC) (250 nM) (Sigma-Aldrich), for 30 min were employed as donor cells, and H9C2 labelled with Did dye (APC) (1:5000) (supplied by Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min (a dye that does not spread throughout cells) were used as acceptor cells. Did dye was used for discerning the two cell types after the dye transfer. In some experiments, in order to demonstrate that Calcein-AM transfer occurs only through the gap junction, NMCs were preincubated with a specific gap junction communication blocker, i.e., carbenoxolone-disodium (CBX) (100 μM) (Sigma-Aldrich), overnight before carrying out the previously reported experimental protocol. All treatments were performed in an atmosphere of 5% CO₂, 95% air at 37 °C in a humidified incubator.
After labelling, cells were co-cultured (NMCs/H9C2 ratio 2:1) for 24 h at 37 °C in a humidified incubator, and APC+FITC+ double-positive cells were analysed through flow cytometry. Cells were acquired with a CyAN ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and analysed by FlowJo^® software (BD Biosciences, V10). At least 80,000 events per sample were collected.