vitro procedures primary normal human lung fibroblasts nhlf  (Lonza)

Journal: Aging (Albany NY)

Article Title: Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

doi: 10.18632/aging.205994

Figure Lengend Snippet: DNA damage is a hallmark of commercial primary IPF fibroblasts in vitro , but not Cdkn2a /p16 ink4a expression or a fibrotic secretome . ( A ) Primary NHLF and IPF cells ( n = 3 different donors, each with 2 technical replicates) were cultured at the same density overnight on standard tissue culture plates in low-serum growth medium and were subsequently fixed. Immunocytochemistry was performed to fluorescently label nuclei (blue) and p21 Waf1/Cip1 (orange). Images were acquired with a 40X water objective using an Operetta High Content Screening instrument and intensity of nuclear p21 Waf1/Cip1 was quantified and calculated as a positive percentage of each population. ( B ) NHLF and IPF cells were treated and imaged as in ( A ), and immunocytochemistry was performed to fluorescently label nuclei (blue) and DNA damage via nuclear γH2A.X (Ser139) foci (orange). The number of nuclear foci per cell was quantified and cells with one or more were reported as positive. ( C ) Following overnight culture in standard tissue culture plates, NHLF and IPF cells were lysed, and qPCR was performed. Expression of senescence and fibrosis-related matrix and secreted factor genes was assessed, and data were normalized to β2m housekeeper expression using the 2 −ΔΔCt method versus NHLF cells. ( D ) Following overnight culture as in ( C ), cell culture supernatants were collected and assayed for determination of the concentration of common fibrosis-related secreted proteins by MSD kits. Statistical analysis was performed using an unpaired t -test ( A , B ) or a two-way ANOVA with a Bonferroni post-test ( C , D ) in GraphPad Prism: * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars represent standard error of the mean (SEM).

Article Snippet: Primary normal human lung fibroblasts (NHLF) were purchased from Lonza and ATCC, and diseased IPF lung fibroblasts were purchased from BioIVT, Lonza, or ATCC.

Techniques: In Vitro, Expressing, Cell Culture, Immunocytochemistry, High Content Screening, Concentration Assay

Journal: Aging (Albany NY)

Article Title: Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

doi: 10.18632/aging.205994

Figure Lengend Snippet: Secreted factors from senescent alveolar epithelial cells potentiate pro-fibrotic gene expression and protein secretion more robustly than direct damage of fibroblasts. ( A ) Schematic summarizing the experimental protocol of direct damage of NHLFs with bleomycin or indirect treatment of NHLFs by transferring conditioned medium from bleomycin-treated AECs. Image created with https://www.biorender.com/ . ( B ) Treated or untreated NHLF cells were lysed, and qPCR gene expression analysis was performed. Expression of senescence and fibrosis-related matrix and secreted factor genes was assessed, and data were normalized to β2m housekeeper expression using the 2 −ΔΔCt method. ( C ) Senescent or non-senescent AEC conditioned medium or blank AEC medium was diluted 1:3 in fibroblast medium and incubated on NHLFs for 5 days. Cells were lysed for gene expression analysis of senescence and fibrosis-related genes. ( D ) Supernatants were collected from the treatments described in C and assayed for fibrosis-related secreted proteins by MSD kits. Data are representative of three independent experiments accounting for 3 biological donors of AECs and 2 donors of NHLFs. Statistical analysis was performed using a two-way ANOVA and a Dunnett’s Multiple Comparisons post-test vs. control conditions in GraphPad Prism: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant. Error bars represent SEM of n = 2 technical replicates.

Article Snippet: Primary normal human lung fibroblasts (NHLF) were purchased from Lonza and ATCC, and diseased IPF lung fibroblasts were purchased from BioIVT, Lonza, or ATCC.

Techniques: Expressing, Transferring, Incubation, Control

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Research strategy. (a) Fibroblast activation is understood to be a result of many microenvironmental cues, such as cell–cell communication, fibrillar structure, biochemical cues, and matrix stiffness, which can either drive healthy wound healing or persistence and fibrosis. There is a need for innovative tools for understanding why certain signals drive homeostasis vs disease. (b) Simplified gene cassettes depict the two key genes for fluorescent reporting: a red fluorescent protein expressed under the constitutive PGK promoter and a green fluorescent protein conditionally expressed with the activation of the ACTA2 promoter associated with alpha smooth muscle actin expression. (c) Fibroblasts are transduced at a high titer to create reporter cell populations, where some cells receive more gene cassette copies than others, but always at a one-to-one ratio of red and green fluorescence. (d) Transduced cells then express constitutive DsRed-Express2 and conditionally express ZsGreen, where the one-to-one ratio of red and green genes delivered to each cell allows for normalization by red fluorescence intensity. (e) To expand the reporter system to track temporal fibroblast behavior, three versions of the gene cassette were utilized, where Intermediate and Fast are modified to degrade on different timescales.

Article Snippet: Primary normal human lung fibroblasts (NHLFs, 52-year-old male, Caucasian) were purchased from Lonza.

Techniques: Activation Assay, Expressing, Fluorescence, Modification

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Comparison of αSMA expression with immunostaining assessment. (a) Stable reporter lung fibroblasts (CCL151, male healthy; CCL210, female healthy; and CCL134, female IPF) were cultured on glass for 48 h, fixed, and stained for alpha smooth muscle actin. (b) Quantification of immunostained images demonstrates that samples from all reporter lines (Stable, Intermediate, and Fast) of all cell types had 100% of the cells positive for diffuse staining for alpha smooth muscle actin (n = 3 wells per condition, >50 cells per well). (c) To further differentiate proto-myofibroblasts from mature myofibroblasts, cells with organized αSMA fibers (CCL151-stable reporter) were visually assessed and (d) quantified across all reporter cell lines (n = 3 well per condition, >50 cells per well). (e)–(g) Reporter intensity normalized on a per cell basis was compared for cells with and without organized αSMA fibers for CCL151 (e), CCL210 (f), and CCL134 (g) cell lines. (per cell line, data from all wells were pooled for distributions). Scale bars are 200 μ m. Statistical comparisons in (d) were assessed by one-way ANOVA and comparisons in (e)–(g) were assessed by Welch's t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Primary normal human lung fibroblasts (NHLFs, 52-year-old male, Caucasian) were purchased from Lonza.

Techniques: Comparison, Expressing, Immunostaining, Cell Culture, Staining

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Comparison of reporters between cells of different tissue origin. ZsGreen(+) population fractions for CCL151 (male healthy lung fibroblasts), CCL210 (female healthy lung fibroblasts), and CCL134 (female IPF lung fibroblasts) Stable, Intermediate, and Fast reporters after 48 h culture were assessed by flow cytometry (initial seeding density 5000 cell/cm 2 ). Statistical differences were determined within reporters by one-way ANOVA. Error bars represent standard deviation, n ≥ 3 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Primary normal human lung fibroblasts (NHLFs, 52-year-old male, Caucasian) were purchased from Lonza.

Techniques: Comparison, Flow Cytometry, Standard Deviation

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Impact of culture density on the reporting of fibroblast activation. CCL151, CL210, and CCL134 lines at different initial seeding densities and culture timepoints were investigated by flow cytometry with (a) Stable, (b) Intermediate, and (c) Fast reporters. Data for 5000 cells/cm 2 48 h conditions are repeated here from for ease of comparison. Statistical differences were assessed by one-way ANOVA within each cell line and reporter. Error bars depict standard deviation with n ≥ 3 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Primary normal human lung fibroblasts (NHLFs, 52-year-old male, Caucasian) were purchased from Lonza.

Techniques: Activation Assay, Flow Cytometry, Comparison, Standard Deviation

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Mechanical properties of the culture substrate impact extent of fibroblast activation. (a) Workflow for assessing the impact of culture surface mechanical properties on αSMA reporting behavior; CCL151 fibroblasts of all reporter types (Stable, Intermediate, and Fast) were cultured on surfaces with different stiffness and assessed using flow cytometry. Error bars represent standard deviation of n ≥ 3 replicates. (b) Stable, (c) Intermediate, and (d) Fast reporter ZsGreen(+) population fractions after culture on soft (E∼2 kPa) and stiff (E∼50 kPa) substrates over two passages. (e) Stable, (f) Intermediate, and (g) Fast box-cox transformed (λ = 0.075) distributions of normalized reporter intensities for individual cell events, where arrows point to shifts in population from P1 to P2. Statistical analysis in panels (b)–(d) were assessed by two-way ANOVA, with passage and culture substrate as factors. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Primary normal human lung fibroblasts (NHLFs, 52-year-old male, Caucasian) were purchased from Lonza.

Techniques: Activation Assay, Cell Culture, Flow Cytometry, Standard Deviation, Transformation Assay

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Transduction of primary cells with Stable reporter lentivirus. (a) Population percentage of transduced NHLFs expressing DsRed, indicating successful transduction. Error bars depict the standard deviation of 3 technical replicates. (b) Flow cytometry histogram shifts of NHLFs after transduction. (c) Comparison of NHLF (MOI 6) ZsGreen(+) population and cell lines (data previously shown in ). Error bars represent the standard deviation of n ≥ 3 technical replicates, with >3 biological replicates for cell lines and 1 biological replicate for primary NHLFs. (d) Fluorescence imaging of MOI 6 NHLFs at day 4 after transduction. Scale bars are 200 μ m.

Article Snippet: Primary normal human lung fibroblasts (NHLFs, 52-year-old male, Caucasian) were purchased from Lonza.

Techniques: Transduction, Expressing, Standard Deviation, Flow Cytometry, Comparison, Fluorescence, Imaging

Journal: bioRxiv

Article Title: Dextromethorphan inhibits collagen transport in the endoplasmic reticulum eliciting an anti-fibrotic response in ex-vivo and in vitro models of pulmonary fibrosis

doi: 10.1101/2023.04.19.537530

Figure Lengend Snippet: A, immunofluorescence staining against ECM deposited COL1 in NHLFs upon TGFb1 and TGFb1+DXM treatment. DXM used in this assay was obtained from a different commercial source. B, quantification of ECM deposited COL1 (immunofluorescence) by different cell types upon in-vitro culture. Normal Human Lung Fibroblasts (NHLFs), BJ-5ta, hTERT-immortalized foreskin fibroblasts, IPF-LF, Lung fibroblasts obtained from idiopathic pulmonary fibrosis patient, Pericytes, immortalized kidney pericytes. C, label-free SHG imaging of ECM deposited fibrillar collagen by NHLFs after 7 days of in-vitro culture (TGFb1 and TGFb1+DXM). SHG images represent fibrillar collagen signal, while AF represents 2-photon excited autofluorescence that gives an estimate of number of cells in the image field of view. D, quantification of SHG signal in each image normalised to AF, with subsequent normalization to the SHG signal from TGFb1 stimulated NHLFs. E, quantification of varying concentration of DXM on ECM deposited COL1 (immunofluorescence) in in-vitro cultured NHLFs. F, representative images (sum projections) of regions of interest from label-free SHG signal acquired from hPCLS (5mm in diameter and 350µm thickness). The hPCLS were stimulated with DMSO, TGFb1+MMPi and TGFb1+MMPi+DXM. Scale bar = 500µm. G, quantification of SHG signal across respective conditions. Color of the dot represents patient ID. Each dot represents a ROI analysed in respective hPCLS of the patient. 3-4 hPCLS were imaged per patient per condition. Patient n= 4. One-way ANOVA was used for testing statistical significance. p-value ≤0.001****. Note: FDA screen image analysis was performed on two replicates of experiments, while all other analysis in the figure here had n=3. Students t-test was used for testing statistical significance. p-value ≤0.01**, p-value ≤0.001***.

Article Snippet: Using cell seeder (ThermoFisher, Multidrop Combi #5840300) primary normal human lung fibroblasts were plated at a density of 6000 cells per well of a 96 well glass bottom plate (Zellkontakt #5241).

Techniques: Immunofluorescence, Staining, In Vitro, Imaging, Concentration Assay, Cell Culture

Journal: bioRxiv

Article Title: Dextromethorphan inhibits collagen transport in the endoplasmic reticulum eliciting an anti-fibrotic response in ex-vivo and in vitro models of pulmonary fibrosis

doi: 10.1101/2023.04.19.537530

Figure Lengend Snippet: A - B, representative immunofluorescence staining acquired using a confocal microscope. The images represent single optical planes from the respective z-stacks. Nuclei were stained using Hoechst 33342, primary antibodies against HSP47, COL1 and TANGO (TANGO1) were used to visualize the respective endogenous proteins in NHLFs (TGFb1 and TGFb1+DXM stimulated). N=2 (number of time the experiment was repeated) for each immunostaining experiment. Scale bar= 50µm. C-D, representative confocal images of NHLF stained for COL1 and TANGO1 (TANGO) upon treatment with DMSO and DXM, B, after 24 hours wash out of the DXM drug. N=2. Inset zooms represent areas in dashed square boxes from merge column of images. Scale bar= 50µm.

Article Snippet: Using cell seeder (ThermoFisher, Multidrop Combi #5840300) primary normal human lung fibroblasts were plated at a density of 6000 cells per well of a 96 well glass bottom plate (Zellkontakt #5241).

Techniques: Immunofluorescence, Staining, Microscopy, Immunostaining