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.