Journal: Cell reports

Article Title: Interferon (IFN)-γ promotes monocyte-mediated lung injury during influenza infection

doi: 10.1016/j.celrep.2022.110456

Figure Lengend Snippet: Key Resources Table

Article Snippet: Primary Bronchial/Tracheal Epithelial Cells; Normal, Human , ATCC , Cat#PCS-300-01.

Techniques: Virus, Recombinant, cDNA Synthesis, SYBR Green Assay, Protein Extraction, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Software, Microscopy, Flow Cytometry, Fluorescence

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Electronic Nicotine Delivery System Aerosol-induced Cell Death and Dysfunction in Macrophages and Lung Epithelial Cells

doi: 10.1165/rcmb.2019-0200oc

Figure Lengend Snippet: Figure 1: ENDS aerosol exposure causes increased cell death in human lung epithelial cells The BEAS-2B cell line was exposed to ENDS aerosol with (PG/VG N+) and without nicotine (PG/VG N-), 100μM Nicotine (Nicotine), HEPA filtered air (Air), and room temperature (Untreated) for 4 min. Cell death was analyzed 24h after treatment by flow cytometry using Caspase-3/7 stain and SYTOXTM permeability dye. Representative plots are shown in (A) and quantification show in (B-E). Necrotic cells are defined as SYTOXTM +, Caspase-3/7- (i) (B). Secondary necrotic cells are dual positive (ii) (C). Apoptotic cells are Caspase-3/7+, SYTOXTM- (iii) (D). All cells with activated Caspase-3/7 (E). Experiments were performed 3 times with an n > 3 per group. *=p<0.05 compared to untreated, #=p<0.05 compared to air, &=p<0.05 compared to PG/VG N-, %=p<0.05 compared to Nicotine. Data were analyzed using ANOVA followed by Tukey’s multiple comparisons test. Mean values are shown with SEM.

Article Snippet: Primary human bronchial epithelial cells (ATCC® PCS-300-010TM) were cultured on 24-well transwell inserts and grown in PneumaCultTM Medium system (Stemcell Technologies Inc.).

Techniques: Aerosol, Flow Cytometry, Staining, Permeability

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Electronic Nicotine Delivery System Aerosol-induced Cell Death and Dysfunction in Macrophages and Lung Epithelial Cells

doi: 10.1165/rcmb.2019-0200oc

Figure Lengend Snippet: Figure 2: ENDS aerosol exposure causes increased cell death in mouse lung epithelial cells The MLE12 cell line was exposed to ENDS aerosol with (PG/VG N+) and without nicotine (PG/VG N-), 100μM Nicotine (Nicotine), HEPA filtered air (Air), and room temperature (Untreated) for 4 min. Cell death was analyzed 24h after treatment by flow cytometry using Caspase-3/7 stain and SYTOXTM permeability dye. Representative plots are shown in (A) and quantification show in (B-E). Necrotic cells are defined as SYTOXTM +, Caspase-3/7- (i) (B). Secondary necrotic cells are dual positive (ii) (C). Apoptotic cells are Caspase-3/7+, SYTOXTM- (iii) (D). All cells with activated Caspase-3/7 (E). Experiments were performed 3 times with an n > 3 per group. *=p<0.05 compared to untreated, #=p<0.05 compared to air, &=p<0.05 compared to PG/VG N-, %=p<0.05 compared to Nicotine. Data were analyzed using ANOVA followed by Tukey’s multiple comparisons test. Mean values are shown with SEM.

Article Snippet: Primary human bronchial epithelial cells (ATCC® PCS-300-010TM) were cultured on 24-well transwell inserts and grown in PneumaCultTM Medium system (Stemcell Technologies Inc.).

Techniques: Aerosol, Flow Cytometry, Staining, Permeability

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Electronic Nicotine Delivery System Aerosol-induced Cell Death and Dysfunction in Macrophages and Lung Epithelial Cells

doi: 10.1165/rcmb.2019-0200oc

Figure Lengend Snippet: Figure 3: One week of ENDS aerosol exposure causes decreased trans-epithelial resistance, dissociation of cells, and cell death Primary human bronchial epithelial cells were differentiated on transwell inserts into a pseudostratified epithelial layer. These cells were exposed to ENDS aerosol with (PG/VG N+) and without nicotine (PG/VG N- ), HEPA filtered air (Air), 100uM nicotine (Nicotine), and room temperature (Untreated) for 4 min. Trans- epithelial resistance was measured using a WPI EVOM TEER meter and STX3 electrode. Measurements were taken in triplicate and averaged. Background readings of empty wells as well as baseline measurements of each sample were used to calculate the change in resistance over the course of the treatment. Dissociated and dead cells were detected using trypan blue exclusion and counting on a hemocytometer. Treatments occurred once per day for 7 days. Experiments were performed 2 times with an n > 3 per group. *=p<0.05 compared to untreated, #=p<0.05 compared to air, %=p<0.05 compared to Nicotine. Data were analyzed using ANOVA followed by Tukey’s multiple comparisons test. Mean values are shown with SEM.

Article Snippet: Primary human bronchial epithelial cells (ATCC® PCS-300-010TM) were cultured on 24-well transwell inserts and grown in PneumaCultTM Medium system (Stemcell Technologies Inc.).

Techniques: Aerosol

Journal: Journal of Innate Immunity

Article Title: Midkine Is Part of the Antibacterial Activity Released at the Surface of Differentiated Bronchial Epithelial Cells

doi: 10.1159/000346709

Figure Lengend Snippet: Production of MK by airway epithelial cells in vitro and contribution to bactericidal activity of the air surface liquid. a Primary HBEC were cultured in an ALI for 4 weeks to allow differentiation, in the absence of antibiotics. The medium was changed, the apical side rinsed with buffer, and the cells were incubated for another 24 h, whereafter the underlying medium was collected and the apical surface was rinsed with buffer. The amount of MK was determined by ELISA and the results shown represent the mean and SD from three separate experiments. b HAEpiC, primary HBEC, the bronchial epithelial cell lines BEAS-2B and 16-HBE, and the alveolar epithelial cell line A549 were grown to near confluence in a flask. The cell medium was changed and the cells incubated for 72 h, the medium collected and the MK content determined by ELISA. No MK could be detected in the medium from the A549 cell line (data not shown). The results represent the mean and SD from three different incubations. c, d To investigate possible bactericidal activity in the air surface liquid, HBEC were grown to confluence and allowed to differentiate using the air-liquid system in the presence of RA. After removal of mucus, the apical surface was rinsed with buffer which was incubated with S. pneumoniae (strain TIGR4). One part was used for the viable count assay and one part was processed for SEM. Bacteria incubated with the rinsing fluid show blebbing and disturbed integrity (d), compared with bacteria incubated in buffer alone (c). e To determine whether MK contributes to the bactericidal activity of the rinsing fluid, MK was immunoprecipitated from one portion of the rinsing fluid while another portion was immunoprecipitated using control antibodies. Thereafter, the rinsing fluids were used to investigate bactericidal activity against S. pneumoniae (strain TIGR4), using the viable count assay. The rinsing fluid depleted of MK showed significantly lower bactericidal activity compared with the rinsing fluid that had been immunoprecipitated with control antibodies, suggesting that MK constitutes a significant part of the bactericidal activity in airway surface liquid. Statistical significance was determined using Student's t test for paired observations. IP = Immunoprecipitation.

Article Snippet: Human pulmonary alveolar epithelial cells (HAEpiC) comprised of type 1 and type 2 pneumocytes (3H Biomedical) were grown in poly- L -lysin (Sigma) coated flasks in alveolar epithelial cell medium with supplements (3H Biomedical).

Techniques: In Vitro, Activity Assay, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: Molecular Therapy

Article Title: Long Non-coding RNA BGas Regulates the Cystic Fibrosis Transmembrane Conductance Regulator

doi: 10.1038/mt.2016.112

Figure Lengend Snippet: BGas and as4 mediated regulation of CFTR . ( a ) A schematic depicting the CFTR locus with transcriptional start sites (TSS) for CFTR , EST BG213071 (BGas), and those primers used to evaluate CFTR expression. The sasRNA target site (as4) in the BGas promoter is also shown along with the two exons making up BGas. ( b ) The effects of BGas over-expression using an exogenously expressed BGas (exBGas) on CFTR expression in 1HAEo- cells. The control (pcDNA3.1) and exBGas transfected cells are shown. ( c,d ) The effects of sasRNA as4 relative to control (pU6M2) on ( c ) BGas and ( d ) CFTR expression in 1HAEo- cells. ( e,f ) The effects of exBGas ( e ) and sasRNA as4 ( f ) on CFTR expression in CFPAC cells. ( g ) Dose-dependent effect of an siRNA targeting the as4 site (siRNA4) on ΔF508- CFTR chloride channel transport function in CF Human Bronchial Epithelial (CFhBE) primary cells (epithelial voltage clamp assay, Ussing chamber). ( h ) The localization of biotin containing BGas at the intergenic locus of CFTR in transfected CFPAC cells contrasted with the lambda biotin control. For b–f , the averages of triplicate treated cultures are shown with the standard error of the means and a P value from a paired T -test, * P < 0.05 and ** P < 0.01.

Article Snippet: Briefly, primary human bronchial epithelial (hBE) cells from a CF patient homozygous for ΔF508- CFTR (Asterand Biosciences, Royston, UK) were plated onto Costar Snapwell (Corning, Corning, NY) tissue-culture inserts coated with collagen type IV, and cultured in Bronchial epithelial growth medium (Lonza, Basel, Switzerland).

Techniques: Expressing, Over Expression, Transfection