Cell lines and cell culture
The Laboratory of Allergic Disease 2 (LAD2) human mast cells were kindly provided by A. Kirshenbaum and D. Metcalfe (NIH, USA). The cells were cultured in StemPro-34 medium supplemented with 10 ml/l StemPro nutrient supplement, 2 mM L-glutamine, 100 ng/ml human stem cell factor (hSCF), and 1:100 penicillin-streptomycin in an atmosphere containing 5% CO2 at 37 ℃. MrgprB2-expressing HEK293 cells and NC-HEK293 cells were constructed by HIV-1-based lentiviral vectors. Both the cells cultured by DMEM. Human mast cell line-1 (HMC-1) was purchased from BLUEFBIO (Shanghai, China).
1 × 106 LAD2 cells were incubated in a 96-well late overnight at 37 ℃ with 5% CO2, then the culture medium was removed. For tacrolimus short-term use, LAD2 cells (and MRGPRX2-knockdown LAD2 cells) were treated with 25-, 50-, 100 uM tacrolimus diluted in Tyrode’s solution for 30 minutes, and 30 μg/ml C48/80 was used to set positive control. For tacrolimus long-term use, LAD2 cells were treated with 0.5 nM tacrolimus or vehicle respectively for 3 d. Then, cells and culture medium were collected.
Cells were used to detect intracellular calcium mobilization assay, western blot and real time qPCR analysis. Moreover, culture medium was used to test β-hexosaminidase release and inflammatory cytokines release.
Small interfering (si)RNA transfection of LAD2 cells
The siRNAs targeting MRGPRX2 leading to specific knockdown or non-specific siRNAs were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). The siRNA sequences were as follows: Negative Control siRNA, forward, 50-UUCUCCGAACGUGUCACGUTT-30, and reverse, 50-ACGUGACACGUUCGGAGAATT-30; MRGPRX2 knockdown siRNA, forward, 50-GUACAACAGUGAAUG GAAATT-30, and reverse, 50-UUUCCAUUCACUGUUGUACTT-30. For transfection, the siRNAs were delivered using Lipofectamine® 2000 transfection reagent in accordance with the manufacturer’s instructions. Cells were then used for the β-hexosaminidase assay, tryptase and cytokines release assay.