Cellamp direct tb green rt qpcr kit

Human adult high-calcium low-temperature (HaCaT) cells (#300493; CLS Cell Lines Service, Eppelheim, Germany) [23 (link)] constitute an immortalized keratinocyte cell line established from the long-term primary culture of human adult skin keratinocytes under specific Ca²⁺ concentrations and temperature conditions. HaCaT cells were cultivated in MG-30 (DMEM, high glucose, ready-to-use, with serum, CLS Cell Lines Service), supplemented with penicillin-streptomycin (Sigma-Aldrich; penicillin (50 units/mL) and streptomycin (50 µg/mL)) at 37 °C, 5% CO₂. In 96-well plates, HaCaT cells (4 × 10⁴ cells/well) were seeded and cultured for 24 h at 37 °C, 5% CO₂. After the initial 24 h, each well received 10 µL of isolated microvesicles (MVs) (with or without polyphenols; protein concentration adjusted to 0.4 µg/µL) and was incubated for 6 h at 37 °C, 5% CO₂. Dulbecco’s PBS (DPBS; Thermo Fisher Scientific) served as the control. A CellAmpTM Direct TB Green RT-qPCR Kit (TaKaRa Bio Inc.) was utilized for cell lysis, reverse transcription reactions, and real-time RT-PCR following the manufacturer’s protocol. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard to normalize mRNA levels among the test samples. The expression level of each gene is presented relative to the expression level in the control. The primer sequences used are presented in Table 1.

HaCaT cells were cultured in MG-30 (DMEM High Glucose ready-to-use, with serum, CLS Cell Lines Service, Eppelheim, Germany) supplemented with 50 units/mL of penicillin and 50 μg/mL of streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C under 5% CO₂. HaCaT cells were seeded at 4.0 × 10⁴ cells/well in a 96-well plate and cultured at 37 °C in 5% CO₂ for 24 h. Isolated MVs (10 µL) were added and incubated at 37 °C in 5% CO₂ for 6 h. For the control, 10 µL of Dulbecco’s PBS (Thermo Fisher Scientific, Waltham, MA, USA) was used instead of MVs. Cell lysis and reverse transcription reactions were performed using the CellAmp^TM Direct TB Green RT-qPCR Kit (TaKaRa Bio Inc., Shiga, Japan). The real-time PCR reaction was performed using Thermal Cycler Dice^® Real-Time System II (TaKaRa Bio Inc., Shiga, Japan). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard to correct differences in the amount of mRNA between samples. The fold change value with a logarithm transformation [log 2 (2^–ΔΔCt)] was calculated. The sequences of the primers are shown in Table 2.

RNA was extracted using a RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA), and real-time RT-PCR analysis was performed using SYBR Green I (Roche Diagnostics, Basel, Switzerland) on a LightCycler^® 96 (Roche Diagnostics). Primer sequences and annealing temperatures are provided in Table 1. Gene expression data were normalized to the housekeeping gene encoding hypoxanthine phosphoribosyltransferase (HPRT) 1 and expressed as a ratio of the values obtained for WT mice and control RAW264.7, respectively.
The CellAmpTM Direct TB Green RT-qPCR Kit (TAKARA Bio, Shiga, Japan) was used for primary cultured stellate cells according to the manufacturer’s protocol. Gene expression data were normalized to hypoxanthine phosphoribosyltransferase 1 and expressed as a ratio of the values obtained for the CM from PBS-treated-RAW264.7 cells in addition to LPS (Sigma-Aldrich, St. Louis, MO, USA).