Iscript cdna synthesis kit

Bioprinted Alg-PBA-Ca hydrogel and alginate hydrogel encapsulated with mouse chondrocytes were first depolymerized by a depolymerizing solution (Cell Applications Inc., San Diego, CA, USA) to remove Ca²⁺ crosslinking; this was followed by homogenization with a bead mill homogenizer (Fisher Scientific, Waltham, MA, USA) in the cell lysis buffer (Qiagen, Hilden, Germany) to release the cytoplasm. The total RNA was extracted from the cell lysate with the RNeasy mini-kits (Qiagen) following the protocol. The extracted total RNA was transcribed into complementary DNA (cDNA) with an iScript cDNA synthesis kit (Quantabio, Berverly, MA, USA). Real-time PCR analysis was conducted in a StepOnePlus™ Real-Time PCR System (Thermo Scientific, Waltham, MA, USA) with SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) dye. The cDNA samples were evaluated for the target genes and the housekeeping gene (Actb). The primer sequences are shown in Table S1. The relative expression of each studied gene was determined by the comparative Ct (2 ^−ΔΔCt) method [45 (link)]. Each group had three replicates.

Trizol RNA extraction buffer was used to isolate RNA isolation of SH-SY5Y wt and SH-SY5Y GH+ cells. According to the manufacturer’s instructions, RNA reverse-transcribed to cDNA using the iScript cDNA Synthesis Kit (Quantabio, Beverly, MA, USA). Then, 1000 ng of cDNA was used for PCR reaction. Expression of GH (Gene ID: 2688) was determined by forward and reverse primers against the GH gene; 5′ AGGATCCCAAGGCCCC-3′,5′-TAGAAGCCACAGCTGCCCTCCAC -3′, respectively. Expression of 18S (Gene ID: 6222) was determined by forward and reverse primers against the 18S gene 5′-GCAGAATCCACGCCAGTACAAG-3′ and 5′-GCTTGTTGTCCAGACCATTGGC-3′, respectively.

Alcian blue 8GX (Alfa Aesa, Karlsruhe, Germany), bicinchoninic acid (BCA) (Sigma Aldrich, Munich, Germany), Chelex^® 100 Resin (Bio-Rad, Hercules, CA, USA), 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) (Carl Roth, Karlsruhe, Germany), Dulbecco’s Modified Eagles Medium (DMEM) (PAN-Biotech, Aidenbach, Germany), Fetal calf serum (FCS) (CCPro, Oberdorla, Germany), iScript cDNA Synthesis Kit (Quantabio, Beverly, MA, USA), non-essential amino acids (NEAA) (Sigma Aldrich, Munich, Germany), NucleoSpin II (Macherey-Nagel GmbH & Co. KG, Berlin, Germany), pararosaniline hydrochloride (TCI, Eschborn, Germany), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma Aldrich, Munich, Germany), sulforhodamine B (SRB) (Sigma Aldrich, Germany), SYBR™-Green (Quantabio, Beverly, MA, USA), ZnSO₄x7H₂O (Sigma Aldrich, Munich, Germany). All other chemicals were purchased from standard sources.