Tb green premix taq

Cells were cultured as described above. The cell culture supernatant was removed and the cells were washed twice with 2 mL ice cold PBS. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was added to the cells and total RNA was extracted using PureLink RNA Mini Kits. The amount of RNA was quantified using a NanoDrop 1000 (ND-1000; Thermo Scientific, Rockford, IL) spectrophotometer. cDNA was synthesized using PrimeScript RT reagent kits (Perfect Real Time; Takara Bio, Shiga, Japan). The mRNA expression of CTL1, TNF-α, IL-6, MMP9, and β-actin were determined using TB Green Premix Taq (Takara Bio). Primer sequences are shown in S2 Table. Real-time PCR was performed using a LightCycler 96 instrument (Roche Diagnostics GmbH, Mannheim, Germany). The mRNA of the target genes were normalized to those of β-actin.

DMEM high glucose medium (lot number: 201809) was purchased from gibco; Sijiqing fetal bovine serum (lot number: 20180325) was purchased from Hangzhou Tianhang Biotechnology Co., Ltd.; (PBS) Phosphate buffer (batch number: 20180928), penicillin-streptomycin mixed solution (batch number: 201803), 0.25% trypsin digestion solution (batch number: 201710), EDTA-free trypsin digestion solution (lot number: 201901) were purchased from Solarbio, USA; Enhanced CCK-8 reagent was purchased from Shanghai Shangbao Biotechnology Co., Ltd.; (DMSO) dimethyl sulfoxide (lot number: EZ1609C224) was purchased from Biofroxx; AnnexinV-FITC/PI apoptosis double staining kit (lot number: 20181217), 1 × Binding Buffer (lot number: 20190114) were purchased from Jiangsu KGI Bio; Homo-BAX-R, Homo-BAX-F, Homo-Bcl2-R, Homo-Bcl-2-F, Homo-CASP3-R, Homo-CASP3-F, Homo-ACTB-R, Homo-ACTB-F were purchased from Shanghai Shenggong Synthetic Company; RNA extraction kit (article number: 9767, batch number: 20181124), reverse transcription kit (article number: RR036A), TB Green Premix Taq (article number: RR820) were purchased from TAKARA; absolute ethanol (article number: 64–17-5) was purchased from Guangzhou Chemical Reagent Factory; 25 cm air-permeable culture flask, 96-well plate, 6-well plate, and cryotube were purchased from corning; 0.22 μM syringe filter was purchased from Millipore.

Total RNA was isolated from fifteen embryos using TRIzol reagent and reverse-transcribed to cDNA using M-MLV reverse transcriptase (Takara, Shiga, Japan). qRT-PCR was carried out using TB Green Premix Taq (Takara, Shiga, Japan) with a real-time PCR detection system (Bio-Rad, Hercules, CA, USA). All primer sequences of target genes are listed in the Table S3. The expression level of a particular gene transcript was calculated based on the standard curve and normalized by the β-actin levels, as no expression changes of β-actin were observed in zebrafish among different treatments. The gene expression levels were calculated by a 2^−ΔΔCT method. The data were reported as fold increases of the control.