Scepter 2

Buffy coats were purchased from the German Red Cross Dresden and PBMC were isolated by density gradient centrifugation, followed by lysis of erythrocytes as previously described [42 (link)]. Monocyte content of the PBMC was analyzed using an automated cell counter (scepter 2.0., Merck Millipore, Darmstadt, Germany). Cells containing 5 × 10⁵ monocytes were seeded into 48 well plates and supplemented with α-MEM containing 10% heat inactivated fetal calf serum (Corning, a trademark of Thermo Fisher Scientific, Waltham, MA, USA), 2 µM L-glutamine and 100 U/mL penicillin and 100 µg/mL streptomycin (all from Gibco, a trademark of Thermo Fisher Scientific, Waltham, MA, USA) (PS) (adhesion medium). After one day of cultivation the medium was changed to α-MEM, 5% heat inactivated FCS, 5% human serum, L-Glu, PS, 25 ng/mL MCSF and 50 ng/mL RANKL (both from Peprotech, Hamburg, Germany) (osteoclast differentiation medium). Cu²⁺ containing medium was prepared by adding different volumes of a 3 mM stock solution of Cu(NO₃)₂ (Sigma-Aldrich, Munich, Germany) in water to the respective medium. For the generation of osteoclasts PBMC were cultivated for 14 days with medium changes twice per week.

The activity of the intracellular dehydrogenases was evaluated by using the Cell Counting Kit-8 (CCK- 8) (Sigma-Aldrich), a highly sensitive test that utilizes water-soluble tetrazolium salt [34 ,39 (link)]. WST-8 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt can be reduced by dehydrogenases in cells, producing an orange colored formazan dye soluble in the culture medium. The cells were counted by using Scepter™ 2.0 (Merck Millipore Corporation, New York, NY, USA) cell counter, seeded in 96-well plates at a density of 8 × 10³ cells/well, and pre-incubated for 24 h under standard conditions. Then, cells were treated for 72 h with different concentrations of the drug solution. Finally, the activity of the intracellular dehydrogenases was evaluated by adding CCK-8 solution (10 μL) to each well and measuring the absorbance at λ = 450 nm after 2 h of incubation with CCK-8 solution, using the microplate reader Victor^® 3V (PerkinElmer^®, Waltham, MA, USA). The changes of the cell viability were expressed as percentage (%) changes of intracellular dehydrogenase activity induced by the drug with respect to the control.

Twenty-four hours before nerve explants were harvested, both AD-MSC and dASCs cells from confluent flasks were collected and counted using a Scepter 2.0 (Merck-Millipore). 100,000 cell per well were plated in 24-well plates (three wells for each time point) and 500 μl of either aMEM or d-aMEM media was used for each well.
Nerve explants were collected from four Sprague Dawley rats as described above, with 15–20 nerve explants collected for each condition. All the nerves were de-sheathed under the microscope and cut into 5–10 mm length. Experimental groups were as follow: D0 healthy nerve explants (D0 c), D3 degenerated nerve explants cultured without cells (D3 c), D3 nerves co-cultured with AD-MSC (uASCs) and D3 nerve explants cultured with dASCs (dASCs).
Using an insert with a 0.4 um porous membrane (Greiner Bio-One), the nerve explants were inserted into the 24 well plate containing the plated cells after a media change and HBSS wash. 1200 μl of DMEM media was added to both the cells and the nerves (across the permeable membrane). The well insert ensured that no direct contact between AD-MSC and explants occurred. The co-cultures were kept in the incubator at 37°C at 5% CO₂ for 3 days without a media change. The nerve explants were collected at each time point and processed for either gene or protein expression as described above.

Cells (10⁵) were grown in the tested conditions and counted after 48 and 96 h using manual cell counter (Scepter 2.0, Millipore, USA), after prior trypsinization with 0.25 % trypsin–EDTA (1×) solution (Gibco Life Technologies, USA).