Rt qpcr kit

Mouse lung tissues were harvested. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), dried by sedimentation and incubated with 50 µl diethylpyrocarbonate-treated water. RNA purity and concentration were determined using an ultraviolet spectrophotometer. Total RNA was reserve transcribed into cDNA using the High-Capacity RNA-to-cDNA™ kit (K1266, Invitrogen; Thermo Fisher Scientific, Inc.). According to the manufacturer's protocol of the RT-qPCR kit (Qiagen GmbH, Hilden, Germany), VDR expression was detected. RNA primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). The following primer sequences were used for the PCR: VDR forward, 5′-GTAAGTACAGGGAGCTATT-3′ and reverse, 5′-GATCTGAATGAAGAAGGCT-3′; GAPDH forward, 5′-AAGATCGAGAATTGACA-3′ and reverse, 5′-GTCGGTGTGAACGGATTTG-3′.

Human lymphocyte separation medium, 4-aminopyridine (4-AP), N-2-hydroxyethylpiperazine-N'-2′-ethanesulfonic acid (HEPES), and K⁺ asparate were obtained from Sigma-Aldrich (St. Louis, MO, USA). The TRIzol reagent, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) solution and the reverse transcription kit (catalog no. K1622) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) kit (catalog no. 204054) was obtained from Qiagen (Hilden, Germany). In addition, the anti-NLRP3 (catalog no. 13158S; 1:1,000), anti-caspase-1 (catalog no. 3866S; 1:1,000), anti-IL-1β (catalog no. 31202S; 1:1,000) and anti-GAPDH (catalog no. 2118S; 1:1,000) rabbit monoclonal antibodies, as well as the anti-rabbit IgG, alkaline phosphatase-linked secondary antibody (catalog no. 7054S, 1:2,000), were from Cell Signaling Technology, Inc. (Beverly, MA, USA). The IL-1β ELISA kit (catalog no. BMS224/2) was obtained from eBioscience (San Diego, CA, USA), while the FITC-labeled anti-CD3 antibody (catalog no. 340571) and recombinant human IL-2 (rIL-2) were from BD Biosciences (San Jose, CA, USA). The RPMI 1640 culture medium and fetal bovine serum were from GE Healthcare (Chicago, IL, USA). The other patch-clamp associated reagents were obtained from Amresco (Solon, OH, USA).