IB3-1 cells (CF cells) are human bronchial epithelial cells derived from patient with CF with a mutant ΔF508/W1282X genotype. The CF phenotype of the IB3-1 cells was corrected in the S9 cell line (non-CF cells; non-CF) by transfection with WT adeno-associated virus expressing CFTR. Both cells were grown on plastic support under liquid-liquid conditions in LHC-8 basal medium (Life Technologies) supplemented with 5% fetal bovine serum.

CuFi-1 (CF cells) and NuLi cells (non-CF) were a gift from J. Zabner (University of Iowa). The CuFi-1 cells were derived from human bronchial epithelia from a patient with CF (CFTR mutant genotype ΔF508/ΔF508), and the NuLi cells were derived from the normal lung of a 36-year-old patient. These cells were grown under liquid-liquid conditions on human placental collagen-coated flasks (Sigma-Aldrich) with bronchial epithelial growth medium (Lonza).

The CF patient–derived cells are represented by polarized mucociliary-differentiated human airway epithelium that was reconstituted in vitro using cells isolated from patient (MucilAir Epithelix CF-202; a 24-year-old female with homozygous ΔF508/F508) and grown on Transwell air-liquid interface. The wt CFTR human primary cells are represented by polarized mucociliary-differentiated human airway epithelium that was reconstituted in vitro with cells isolated from donor (MucilAir Epithelix; MD-801, a 59-year-old female donor with no pathology reported) and grown on Transwell air-liquid interface.

Stable MCU-silencing IB3-1 cell clone was obtained using shRNA targeting MCU (TRCN0000133861) purchased from Sigma-Aldrich. IB3-1 cells were cultured in tissue dish to 75% of confluence and infected with lentiviral-driven shRNA targeting MCU for 48 hours. The infected cells were selected by the addition of 5 μM puromycin to the culture medium, fed every 2 to 3 days with selection medium, and checked for cell death after 3 to 7 days. Resistant cells were normally observed after about 2 weeks of selection. Individual cells were selected randomly and placed in separate wells within a 24-well plate. Clones were grown to confluency and expanded in a maintenance puromycin media (0.5 μM). Expanded clones were retested for MCU expression before any further studies. Primary Nlrp3+/+ and Nlrp3−/− MEFs were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin-streptomycin-glutamine (100×) liquid.

For the transient overexpression experiments, pNEO plasmid encoding a myc-tagged VAPB and a HA-tagged PTPIP51 and ATF5 cloned in pEGFP-N1 were transfected with Lipofectamine LTX (Life Technologies) according to the manufacturer’s recommendations. After 36 hours, the transfected cells were infected with the well-characterized, motile, nonmucoid P. aeruginosa laboratory strains, named PAO1, donated by A. Prince (Columbia University). Bacterial colonies from overnight cultures on trypticase soy agar (Difco) plates were grown in 20 ml of trypticase soy broth (Difco) at 37°C with shaking until an optical density (OD660nm) corresponding to 1 × 107 CFU/ml was reached. The bacteria were washed twice with Krebs-Ringer buffer (KRB), and diluted in each specific serum-free medium before infection. PAO1 was added to cells as indicated in the captions for Figs. 1 through 5.

Mucopurulent material was harvested from the lumens of excised human CF lungs infected with P. aeruginosa at the UNC Adult Cystic Fibrosis Center. SMM from several patients was pooled to assure homogeneous stimulus throughout the experiments. Ten microliters of SMM (1:100) or KRB (a control for SMM) was applied to cells for 6 hours.

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