Sybr green supermix

RNA from cultured cells was extracted and purified using Nucleo Spin RNA II columns (Machery-Nagel Inc., Easton, PA USA). First strand cDNA was synthesized from RNA using random hexamer and oligo dT priming and moloney murine leukemia virus reverse transcriptase (MMLV, Life Technologies) to produce random primed cDNA. Quantitative PCR measurements were performed using the SA Bioscience SYBR Green Supermix (QIAGEN, Valencia, CA USA) reagents on an iCycler Real-Time PCR Detection System (Bio-Rad Inc., Hercules, CA USA). Each reaction was carried out in 15 μl reaction volumes of SA Bioscience SYBR Green Supermix with 5 pM of each primer and diluted first-strand cDNA. All cDNA samples were measured in duplicate. Ribosomal protein large protein 38 (Rplp38) was used as the housekeeping gene control for ΔC_T calculation [ΔC_T  =  (C_T of the target gene) – (average C_T of housekeeping genes)]. Primer sequences were listed in Table S2. Fold change expression values were calculated using 2^−ΔΔCT methods[57] (link), where ΔΔC_T  =  (ΔC_T of the treatment sample) – (ΔC_T of control treatment samples) with no added IGF1 as control treatment and normalized to 1.

Total RNA were isolated from indicated cells as described as above. Single-strand cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., United States). Primer sets for IGFBP2, GREM1 (Gremlin 1, DAN Family BMP Antagonist), BARX1 (BarH-like homeobox 1), and ALPL (alkaline phosphatase, liver/bone/kidney) were purchased from the Integrated DNA Technologies (IDT, Coralville, Iowa., United States). Other primer sets were synthesized by IDT as follows: TFAP2A(transcription factor AP-2 alpha)-For: 5′-GAGCCATGGCACGCACGAGACGGTATCTA-3′, TFAP2A-Rev: 5′-GAGCTCGAGCTCGCAGTCCTCGTACTTGA-3′; LYPD6B (LY6/PLAUR Domain Containing 6B)-For: 5′-GTTTCCTGACCCGTGAAATG-3′, LYPD6B-Rev: 5′-GTCCCGTCCAGATGTTGG-3′; RUNX2-For: 5′-TTACTTACACCCCCCAGTC-3′, RUNX2- Rev: 5′-CACTCTGGCTTTGGG AAGAG-3′;and endogenous control GAPDH-For: 5′-AGGTCGGAGTCA ACGGATTTG-3′, GAPDH-Rev: 5′-GGGGTAACTGTGC-CTATTCG-3′. Quantitative PCR using SYBR Green SuperMix (Qiagen) was performed as we previously described (Lu et al., 2014 (link)). The level of mRNA expression was measured using threshold cycle (CT) according to the ΔΔCT method (Livak and Schmittgen, 2001 (link)).

Real-time PCR profiling of mRNAs was conducted with the SYBR Green-based RT² Profiler PCR Array System (Qiagen). Briefly, total RNA was extracted, and first-strand cDNA was synthesized using the RT² First Strand Kit (Qiagen). A PCR primer assay was performed using SYBR Green Supermix (Qiagen) and gene-specific primers that attached to the bottom of the mouse phagocytosis array panel in the CFX96 Real-Time PCR Detection System (Bio-Rad). PCR primer assay data were analyzed on the Qiagen analysis website (www.qiagen.com/us/shop/genes-and-pathways/data-analysis-center-overview-page/), and the scatter plot result was the output.

LD, HD, or LDHD designated for inflammatory induction was primed or not by incubation in culture media containing 10 ng/ml IFNγ and 15 ng/ml TNFα (R&D Systems) for 48 h, as previously performed (Kouroupis et al., 2019 (link); Bowles et al., 2020 (link)). Cell supernatants were collected, centrifuged, and analyzed for the release of pro-inflammatory and anti-inflammatory factors. Cells were then detached and counted, and pellets were collected for gene expression analysis by using RT2 Profiler Arrays designed for Mesenchymal Stem Cell genes (Qiagen).
Master mixes containing 500 ng/ml of each cDNA sample, SYBR Green Supermix (Qiagen), and ultrapure water were prepared for each sample. Pathway-focused gene expression analysis was pre-assembled with primers of 84 genes related to known mesenchymal stem cells transcription profiles and five housekeeping genes (Supplementary table 2). Ct values were obtained and analyzed by the Qiagen’s Data Analysis Center. βACTIN gene was selected after being tested with reference genes stability test (Ragni et al., 2019 (link)) as the optimal housekeeping gene and used to obtain the dCt values.