Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
Solutions
2.2. Cell Growth and Differentiation Protocol
2.3. RNA Extraction, RNA-Sequencing, and Real-Time Quantitative PCR Analysis
2.4. Protein Extraction, Quantification, and Western Blot (WB)
2.5. Histological and Immunofluorescence Staining
2.5.1. Histology
2.5.2. Immunofluorescence Staining
2.6. Intracellular pH
2.7. Short-Circuit Current Measurements in Ussing Chamber
2.8. ASL pH
2.9. Data Analysis
2.9.1. RNA-Sequencing Analysis
2.9.2. Airway Epithelial Cell Subtype Identification
2.9.3. Patterns of Gene Expression and Signaling Pathways Using NetworkAnalyst
2.9.4. Statistical Analysis of Other Data
3. Results
3.1. UNC and SC Media Induce Distinct Epithelial Phenotypes in CF and NCF Epithelia
3.2. CF Cells are More Susceptible to Changes in Growth Media than NCF Cells
3.3. Growth Conditions Dictate Airway Epithelial Cell Identity
3.4. Pathway Analysis Reveals a Differentiation Medium Dependent Shift in Expression of Genes Involved in Ion and Fluid Transport Homeostasis
3.5. UNC-Grown CF Cells Express Higher Levels of ATP12A
3.6. CFTR Expression and Function are Altered by Differentiation Media in CF Cells
3.7. Differentiation Media Modulate CF Cells Response to CFTR Modulators
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Target Gene | Primer | Sequence | Product Length |
---|---|---|---|
18S rRNA | Forward | 5′-CTCTAGATAACCTCGGGCCG-3′ | 209 |
Reverse | 5′-GTCGGGAGTGGGTAATTTGC-3′ | ||
ATP12A | Forward | 5′-GGGGCACACTTGTTCATCTTCTGA-3′ | 128 |
Reverse | 5′-GCAAAACATCAGTGAGCATCCTG-3′ | ||
CFTR | Forward | 5′-AGGAGGCAGTCTGTCCTGAA-3′ | 237 |
Reverse | 5′-CACTGCTGGTATGCTCTCCA-3′ |
Protein Target | Manufacturer (Reference) | Species Raised in | Dilution/Concentration (WB/IF) |
---|---|---|---|
Primary Antibodies | |||
β-actin | Sigma-Aldrich (A5441) | Mouse | 1:5000 (WB) |
CFTR (Cystic Fibrosis transmembrane conductance regulator | CFF Therapeutics (596) | Mouse | 1:3000 (WB) |
1:250 (IF) | |||
ATP12A | Sigma-Aldrich (HPA039526) | Rabbit | 1:1000 (WB) |
1:400 (IF) | |||
MUC5AC | Abcam (ab3649, Cambridge, UK) | Mouse | 1:67 (IF) |
Acetyl-α-Tubulin (Lys40) (D20G3) | Cell Signaling Technology (5335, London, UK) | Rabbit | 1:800 (IF) |
Phalloidin (Alexa Fluor 647) | Thermo Fisher Scientific (A30107) | NA | 1.25 µg/mL |
DAPI (4′,6-diamidino-2-phenylindole) | Sigma-Aldrich (D9542) | NA | 1:1000 |
Secondary Antibodies | |||
Anti-mouse Immunoglobulin (Ig)G, HorseRadish Peroxidase (HRP)-linked | Cell Signaling Technology (7076) | Horse | 1:5000 (WB) |
Anti-rabbit IgG, HRP-linked | Cell Signaling Technology (7074) | Goat | 1:5000 (WB) |
Precision Protein™ StrepTactin-HRP Conjugate | Bio-Rad (161038) | NA | 1:5000 (WB) |
Anti-Rabbit IgG (Alexa Fluor 488) | Thermo Fisher Scientific (A11034) | Goat | 5 µg/mL (IF) |
Anti-Mouse IgG (Alexa Fluor 594) | Thermo Fisher Scientific (A11032) | Goat | 2 µg/mL (IF) |
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Saint-Criq, V.; Delpiano, L.; Casement, J.; Onuora, J.C.; Lin, J.; Gray, M.A. Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells. Cells 2020, 9, 2137. https://doi.org/10.3390/cells9092137
Saint-Criq V, Delpiano L, Casement J, Onuora JC, Lin J, Gray MA. Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells. Cells. 2020; 9(9):2137. https://doi.org/10.3390/cells9092137
Chicago/Turabian StyleSaint-Criq, Vinciane, Livia Delpiano, John Casement, Jennifer C. Onuora, JinHeng Lin, and Michael A. Gray. 2020. "Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells" Cells 9, no. 9: 2137. https://doi.org/10.3390/cells9092137