Dulbecco s phosphate buffered saline

The cells utilised were healthy human neural stem cells (hNSC) H9-derived (a commercial neural stem cell cell-line from Gibco, obtained from a healthy female donor, #N7800100) and human sporadic ALS iPS-derived neural stem cells (a commercial neural stem cell cell-line from iXCells Biotechnologies obtained from a 55 years old Caucasian female sporadic ALS donor without any known mutations in SOD1 or C9ORF72, #40HU-007). Both healthy and ALS hNSC were seeded onto Matrigel-coated surfaces prepared by making 1:30 dilution of Matrigel^® hESC-Qualified Matrix (Corning, #354277) into Knockout™ DMEM/F-12 medium, also known as Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (Gibco, #12660012) incubated for 1 h at room temperature. Cells were maintained with hNSC proliferation medium changing it every day after. Once the desired number of cells was reached, at TP0, cells were washed with sterile DPBS, also known as Dulbecco’s Phosphate Buffered Saline (Lonza, #17-512F), detached with TrypLE™ express enzyme (Gibco, #12604021), counted with a haemocytometer and transferred in the desired density to ultra-low attachment PrimeSurface^® 3D culture spheroid plates (S-Bio, #MS-9096MZ) to form neurospheroids.

Blood obtained from each study volunteer at the Austrian Red Cross Blood Transfusion Service of Upper Austria was used to produce autologous APOSEC according to current GMP guidelines. PBMCs were separated from whole blood samples of the participants by density centrifugation using LSM 1077 (Lymphocyte Separation Medium, Lonza, Switzerland). Removal of LSM was achieved by two washing steps using Dulbecco’s phosphate-buffered saline (Lonza, Switzerland). PBMCs were resuspended in phenol red–free CellGro GMP DC medium (CellGenix, Freiburg, Germany) containing no xenogeneic proteins. A sample was drawn for complete blood count to adjust white blood cells to a concentration of 25 × 10⁶ cells/ml. Irradiation with 60 Gy induced apoptosis of PBMCs. By cultivation of apoptotic PBMCs in CellGro GMP DC medium, release of the secretome was achieved. After incubation for 24 h ± 2 h, cells were removed by centrifugation. The supernatant containing the secretome was sterile filtered at a pore size of 0.22 μm. The adequate production of APOSEC was defined by appropriate secretion of the following important cytokines: interleukin (IL)-8 (0–5214 pg/ml), epidermal growth factor (EGF; 25–226 pg/ml), and transforming growth factor-β (TGF-β; 2575–21732 pg/ml).
Lyophilized culture medium not containing any cells (CellGro, CellGenix, Freiburg, Germany) served as placebo.

Following 24 h of treatment, myoblasts were harvested using Dulbecco’s Phosphate-Buffered Saline (Lonza, Nottingham, UK) and stored at −80 °C. RNA of treated cells was isolated using EZ-RNA isolation kit (Biological Industries, Beit Haemek, Israel) and cDNA was synthesised using iScript cDNA synthesis kit (Bio-Rad, Watford, UK). Real-time qPCR analyses were performed on a StepOnePlus™ real-time PCR system (Applied Biosystems, Warrington, UK) using QuantiNova SYBR Green PCR Kit (QIAGEN, Manchester, UK); 10 ng of cDNA per reaction was used in a total volume of 10 μL PCR reaction mixture. Three amplifications, consisted of an initial denaturation cycle at 95 °C for 2 min, followed by 40 cycles of 20 s at 95 °C (denaturation), 20 s at optimal annealing temperature, and 20 s at 72 °C (extension), were performed for each primer, followed by a melt curve analysis. Threshold cycle for each target gene of interest was normalised to the housekeeping gene 18S (annealing temperature, 55.7 °C), which has been extensively used in muscle [18 (link),19 (link),20 (link)], and analysed using the delta-delta (2^−ΔΔCt) method [21 (link)]. The sequences and annealing temperature of all mitochondrial- and ER stress pathway-associated primers used are provided in Table 1 and Table 2, respectively.