Fast sybr green master mix

The Ti6Al4V powder was purchased from AK Medical Co., Ltd. (Beijing, China). Poloxamer 407 powder was obtained from Bayee Chemical Co., Ltd. (Hangzhou, China). BMP-2, OPG, and antibodies used in immunofluorescence were obtained from Abcam (Cambridge, UK). Low Glucose Dulbecco’s Modified Eagle’s Medium (DMEM), streptomycin double-antibody, and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Paraformaldehyde and phosphate-buffered saline (PBS) were obtained from Coolabar (Beijing, China). Cell Counting Kit-8 (CCK-8) and Calcein-AM/propidium iodide (PI) were supplied by Beyotime Biotechnology (Shanghai, China). The osteogenic medium used for alizarin red dye was obtained from Cyagen (Santa Clara, CA, USA). The Perfect Real-Time RE reagent kit from Takara Bio (Dalian, China), 2× Fast SYBR Green Master Mix from Roche Diagnostics (Basel, Switzerland), and TRIzol reagent (Invitrogen, CA, USA) were used. The Runx-2, OPN, and RANKL antibodies were supplied by Abcam (Cambridge, UK) and DAPI from Solarbio (Beijing, China). The Electrochemical Immunoassay Kit was obtained from Roche-Mannheim (Mannheim, Germany). The tartrate-resistant acid phosphatase (TRAP) staining kit was obtained from Sigma-Aldrich (St. Louis, MO, USA). The ultrapure water used in the study was obtained from a Milli-QA10 filtration system (Millipore, Billerica, MA, USA).

ADSCs were seeded in 48-well plates pre-coated with 100 μL HA or 100 μL HA-EGCG hydrogel at the density of 1×10⁴/well under the oxidative environment as described above. After incubation with chondrogenic induction medium for 14 days, cell samples were collected for real-time PCR analysis. In brief, the samples were ground within TRIzol reagent to extract total mRNA. The concentration of acquired RNA was determined by Infinite 200 PRO NanoQuant Microplate Readers (TECAN). Then the RNA (1 μg) was used for cDNA synthesis. Real-time PCR was performed using 2× Fast SYBR Green Master Mix (Roche Diagnostics, Basel, Switzerland) and ABI StepOnePlus™ Real-Time RCR system (Applied Biosystems). The relative mRNAs expression was calculated by the 2^−ΔΔCt formula. The primer sequences of GAPDH, IL-1β, MMP-13, TNF-α, aggrecan (ACAN), type II collagen (COL-2), and Sex determining region Y box 9 (SOX-9), were listed in Table 1.

The expression of genes involved in osteogenic differentiation was determined by real-time quantitative PCR (RT-qPCR), including Runx-2, Col-1, OCN, and OPN, in the BMSCs at 14 and 21 days after osteogenic induction and bone tissues. The primers sequences are listed in Table 1. Total RNA was collected by TRIzol reagent. Synthesis of cDNA was performed using an Eastep Super Total RNA Extraction Kit according to the manufacturer’s instructions. An A260/A280 value of approximate 2.0 was generally accepted for further analysis. The amplification and performance of RT-qPCR were carried out by adopting 2× Fast SYBR Green Master Mix (Roche Diagnostics, Basel, Switzerland) and detected by a LightCycler 480 (Roche Diagnostics). The relative mRNA expression was normalized by GAPDH and calculated according to the formula of the 2^−ΔΔCT method. For RT-qPCR of the bone, tissues were collected and quickly placed in a mortar precooled with liquid nitrogen and repeatedly ground to powder in liquid nitrogen for RT-qPCR as previously described.

To quantify the level of PD-L1 mRNA in the PD-L1 variant overexpressing cells, cDNA, Fast SYBR green Master Mix (Roche), and primers (PD-L1: forward 5′-TGG​CAT​TTG​CTG​ACG​CAT​TT-3′ and reverse 5′-TGC​AGC​CAG​GTC​TAA​TTG​TTT​T-3′; GAPDH: forward 5′-TGC​ACC​ACC​AAC​TGC​TTA​GC-3′ and reverse 5′-GGC​ATG​GAC​TGT​GGT​CAT​GAG-3′) were mixed. The reaction was performed using the LightCycler 96 system (Roche).