Thermo cdna synthesis kit

For nephrotoxicity assessment, the gene expression in kidney tissues and TCMK-1 cells was analyzed using qPCR. Kidney tissues were collected after sacrificing the mice and immediately frozen at −80 °C. For RNA extraction, 5 mg of kidney tissues was dissected into small pieces. TCMK-1 cells (5 × 10⁵ cells/well in a 12-well plate) were cultured in 1 mL of EMEM for 24 h. The cells were treated with 1000 µM of sodium thiosulfate and various OAA concentrations for 1 h and were subsequently stimulated with 20 µM of cisplatin for 4 h. Total RNA was isolated using the TRIzol Reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s protocol. First-strand complementary DNA (cDNA) was synthesized using a Thermo cDNA synthesis kit (Thermo Scientific). qPCR was performed using a Bio-Rad T100 thermal cycler (Bio-Rad Laboratories) according to the manufacturer’s protocol. The primer sequences are shown in Table 1. The number of cycles was optimized to ensure that the product accumulation was in the exponential range. β-actin was used as an endogenous control for normalization. Experiments were repeated three times.

Stable cell lines were generated to evaluate in vitro binding capacity of anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins. For this purpose, total RNA was extracted from anti-CD3/CD28 activated human PBMCs, and the first-strand cDNA was synthesized by Thermo cDNA synthesis kit (Thermo Scientific, USA). Human PD-1 (hPD-1), CTLA-4 (hCTLA-4), CD28 (hCD28), and 4-1BB (h4-1BB) coding genes were amplified by PCR using specific primers and separately inserted into pCHO1.0 expression vector. As described earlier, these constructs were transfected to CHO-K1 by electroporation, and then stable cell lines were produced by puromycin/MTX strategy. The expression of these proteins on the surface of transfected CHO-K1 cells was screened by flow cytometry using anti-PD-1-PE (BioLegend™, USA), anti-CD28-PE (BD Pharmingen™, USA), anti-CTLA4-PE (BioLegend), and anti-4-1BB-PE (BioLegend™, USA) antibodies.

HEp-2 cells were infected with HSV-1 at an MOI of 0.01 and were treated with R. tanguticum nanoparticles (350 µg/ml) at 6, 12, 18, and 24 h intervals, and total RNA was extracted with TRIzol reagent (Ambion^®, Life Technologies Pty Ltd., Carlsbad, CA) according to the manufacturer’s instructions. The cDNA was synthesized in a reverse-transcriptase reaction by using Thermo cDNA Synthesis Kit (Thermo Scientific, Rockford, IL). The immediate early gene α4 (encoding ICP4), early gene U_L29 (encoding ICP8) and late gene U_S6 (encoding glycoprotein gD) were determined by using a Bio-Rad CFX96 instrument according to a two-step protocol (95°C for 2 min, 45 cycles of 5 s at 95°C, 10 s at 60°C, and 15 s at 72°C). The transcription levels of total RNA in each sample were standardized against the GAPDH gene. The sequences of Primers used were as follows: ICP4 (5’-CGACACGGATCCACGACCC-3’ and 5’-GATCCCCCTCCCGCGCTTCGTCCG-3’);ICP8 (5’-CGACAGTAACGCCAGAAG-3’ and 5’-GGAGACAAAGCCCAAGAC-3’); gD(5’-GCCCCGCTGGAACTACTATG-3’ and 5’-TTATCTTCACGAGCCGCAGG-3’);GAPDH(5’-GGTGGTCTCCTCTGACTTCAACA-3’ and 5’-GTTGCTGTAGCCAAATTCGTTGT-3’). Relative quantification was carried out based on the 2^−ΔΔCT threshold cycle method.