Cells to ct 1 step taqman kit

Seventy-two hours after MCAo, the ischemic hemisphere, contralateral hemisphere and spleen were collected, as was normal bone marrow. The FACS-sorted CD11b⁺Ly6C^lowLy6G⁺ cells were lysed using the cells-to-CT1 Step TaqMan Kit (Thermo Fischer Scientific), according to the manufacturer’s recommendations. The resulting lysate was then used in a one-step, real-time reverse transcription polymerase chain reaction (RT-PCR). Specifically, TaqMan gene expression assay for Nox2 (Cybb, assay No. Mm01287743_m1), CHOP (Ddit3, assay No. Mm00492097_m1), and Nox4 (Nox4, assay No. Mm00479246_m1) were used, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference (assay Mm99999915_g1). The RT-PCR used a 7900HT fast real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). Expression was assessed in triplicate. Relative quantification method 2-[Delta][Delta] Ct (2^{-deltalelta Ct}) was used for normalization of gene expression [18 (link)]. All Ct values beyond 40-cycle was excluded from data analysis. Gene expression was normalized with the GAPDH. The level of the gene expression of the ischemic hemisphere, contralateral hemisphere, and spleen was compared with the level of the gene expression in normal bone marrow and expressed as an n-fold ratio.

SCAP stimulated with LPS (1 μg/mL) for 24 h were lysed using the Cells-to CT™ 1-Step TaqMan® Kit (Thermo Fischer Scientific). The resulting lysate was then used for one-step real-time RT-PCR with a TaqMan® gene expression assay for ALX/FPR2 (assay Hs00265954_m1), and with glyceraldehyde 3-phosphate dehydrogenase as an endogenous reference (assay Hs02786624_g1), using a StepOnePlus™ apparatus (Applied Biosystems). The relative ALX/FPR2 gene expression was determined using a comparative delta-delta cycle threshold method (DDCt) with a control group as a calibrator^{46 (link)}.

To determine differences in Pgc1α activity, mRNA levels of endogenous PCK1 were measured. HEK293T cells (4x10⁴) were grown on 96-well plates using complete DMEM (DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 0.1 mg/mL streptomycin and 100 U/mL penicillin). After 24 hours, cells were transfected using GeneJuice (Novagen) following manufacturer's instructions. 24 hours later, mRNA was extracted and cDNA was obtained using Cells to Ct 1-Step Taqman kit (Thermo Fisher Scientific). PCK1 cDNA was amplified using a human PCK1 probe set (Thermo Fisher Scientific) in an Mx3005P qPCR System (Agilent). GAPDH was used as control. PCK1 was quantified as $\frac{1}{2^{\Delta Ct}}$ ; where Δ_Ct is Pck1_Ct − GAPDH_Ct from each sample.