Cfx96 real time thermal cycler detection system

TRIzol (Ambion; Life Technologies, Carlsbad, California) was used to isolate total RNA from OLC at 7, 14, and 21 days after differentiation and from EXP-21 cells. With 200 ng of RNA per sample, RT-PCR was performed using the SYBR Green One-Step Real-Time RT-PCR Master Mix system (Invitrogen; Grand Island, NY, USA) in the CFX96 Real-Time Thermal Cycler detection system (Bio-Rad; Hercules, CA, USA). Amplification conditions were as follows: 15 min retrotranscription at 50°C, 4 min at 94°C and 40 amplification cycles of 20 s at 94°C, 20 s at 60–62°C, and 20 s at 72°C. Dentinogenic mRNA markers were quantitated using a specific primer set (Table 1); the genes evaluated were osteopontin (OPN), osterix (OSX), alkaline phosphatase (ALP), type I-collagen (COL-I), runt-related transcription factor 2 (RUNX2), dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), and β-Actin (β-ACT). DSPP and DMP-1 as specific odontogenic markers were further evaluated in thawed EXP-21 cells to demonstrate phenotype maintenance after cryopreservation. PCR efficiencies were calculated using LinRegPCR (Academic Medical Center, AMC, Amsterdam, Netherlands), and relative gene quantitation was performed following Schefe's method [14 (link)].

BEAS-2B cells were seeded at 1 to 2 × 10⁵ cells/well in a 6-well plate with K-SFM complete media. Once the cells reached 80% to 90% confluence, the complete media were replaced by supplement-free K-SFM for 18 to 24 h, and then cells were co-treated with 5 uM carvedilol with or without 10 µM B(a)P for 6 h. Total RNA was isolated using a RNeasy mini kit (Qiagen, Hilden, Germany). cDNA was obtained using a high capacity cDNA reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was amplified with Power SYBR™ green PCR master mix (Applied Biosystems) and primers for human CYP1A1 and beta-actin. PCR was performed on a CFX96 real-time thermal cycler detection system (Bio-Rad, Hercules, CA, USA). The data were analyzed based on the 2^−ΔΔCt method using beta-actin as the normalization gene. Primer sequences: (CYP1A1, Forward 5′-TGGTCTCCCTTCTCTACACTCTTGT-3′, Reversed 5′- ATTTTCCCTATTACATTAAATCAATGGTTCT-3′and BETA-ACTIN Forward 5′-ACCAACTGGGACGATATGGAGAAGA-3′; Reverse 5-CGCACGATTTCCCTCTCAGC-3′).

Total RNA was isolated from whole skin tissue using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA). cDNA was synthesized with the High-Capacity cDNA Reverse Transcriptase Kit (Thermo Fisher). cDNA and SYBR Green Supermix (Thermo Fisher) were mixed with primers for mouse IL-6 gene and β-actin (the primer sequences are available upon request). qPCR was performed on a CFX96 real-time thermal cycler detection system (Bio-rad, Hercules, CA, USA) and analyzed with the 2^−ΔΔct with β-actin as the normalization control.

According to the protocols provided by the manufacturer, the skin cultures were homogenized and total RNA was isolated using Qiagen’s RNeasy Kit. cDNA was synthesized with the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Grand Island, NY, USA). RNAs were examined for the expression of 84 genes involved in DNA damage signaling using RT² Profile PCR Array Human DNA Damage Signaling Pathway (Catalog #: PAHS-029a, SABiosciences, Frederick, MD, USA). The PCR cycle condition was as follows: 94 °C for 4 min, followed by 40 cycles of 94 °C for 30 s, 62 °C for 30 s, 72 °C for 35 s, and then kept at 72 °C for 10 min. PCR was performed on a CFX96 real-time thermal cycler detection system (Bio-rad) and analyzed with the 2^−ΔΔCt with GAPDH as the reference gene. An artificial cut-off value of greater than a 20% change between the UV treated versus the untreated control was utilized to determine the UV effects. To examine the carvedilol effects, a cut-off value of greater than 30% change between the carvedilol plus UV group and the UV alone group was selected. Individual RT-qPCR analysis was conducted in the same method using gene specific primers for which sequences are available upon request.

The thermal stability of the mutant ACE2 ectodomain proteins were evaluated by differential scanning fluorimetry as follows. Purified ACE2-His proteins were diluted to 200 μg/ml in PBS and placed in 0.2-mL white PCR tubes (Bio-Rad, TLS0851) at 20 μl/tube. After adding 1 μl/tube of SYPRO™ Orange protein gel stain solution (Invitrogen, S6651) diluted with water at 1:150, the tubes were placed in a Bio-Rad CFX96 thermal cycler Real-Time Detection System. Thermal denaturation curves from 25 °C to 95 °C (ramp rate of 1.27 °C/min at 0.5 °C intervals with an equilibration of 5 sec at each temperature before measurement) were acquired by measuring fluorescence intensities using the FRET channel with excitation from 450 to 490 nm and detection from 560 to 580 nm. All data were exported and plotted in Microsoft Excel and the first derivative approach was used to calculate T_m.

Experiments were carried out as described for high-throughput thermal scanning^{67 (link)}. Specifically, SYPRO Orange dye (Invitrogen) was supplied in dimethylsulfoxide at 5000× the working concentration for PAGE staining. Samples of 20 μL per well were prepared by mixing 1 μL of 200× SYPRO Orange (final concentration 10×) with 19 μL of protein (0.1 mg/mL or 2.1 μM) in PBS. Spectra were obtained on a Bio-Rad CFX96 thermal cycler Real-Time Detection System. Thermal denaturation curves (ramp rate of 1 °C/min at 0.2 °C intervals with an equilibration of 5 s at each temperature before measurement) were acquired by measuring fluorescence intensities using the Förster resonance energy transferchannel with excitation from 450 to 490 nm and detection from 560 to 580 nm. Melting temperatures are reported as the average, ±SD from four independent measurements.