Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632
Abstract
:1. Introduction
2. Results
2.1. Isolation of RLKs and Screening of Culture Media
2.2. Conditional and Reversible Immortalization of RLKs by Y-27632
2.3. Proteome Profiling of RLKs in the Presence and Absence of Y-27632
2.4. Characterization of RLKs
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Primary Culture and Maintenance of RLKs
4.3. Phalloidin Staining
4.4. TUNEL Assay
4.5. Sample Preparation for Mass Spectrometry
4.6. Nano-UPLC-MS/MS and Comparative Proteomic Analysis
4.7. Reverse Transcription Polymerase Chain Reaction (RT-PCR)
4.8. Live-Cell Calcium Imaging
4.9. Cytogenetic Analysis
4.10. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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EpiLife | K-SFM | MTCM | |
---|---|---|---|
Basal media | EpiLife™ medium | Defined keratinocytes SFM | IMDM + 10% FBS + 20% MCDB 153 medium |
Supplements | EpiLife™ defined growth supplement, (10 μM Y-27632) 1 | Defined keratinocyte SFM growth supplement, (10 μM Y-27632) 1 | 10 ng/mL EGF, (10 μM Y-27632) 1 |
Antibiotics | 1% antibiotic-antimycotic, 0.015% gentamicin | ||
Calcium level | 60 µM | <0.1 mM | ~1.4 mM |
References | [26] | [27] | [1,28] |
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Chen, Z.; He, W.; Leung, T.C.N.; Chung, H.Y. Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632. Int. J. Mol. Sci. 2021, 22, 6782. https://doi.org/10.3390/ijms22136782
Chen Z, He W, Leung TCN, Chung HY. Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632. International Journal of Molecular Sciences. 2021; 22(13):6782. https://doi.org/10.3390/ijms22136782
Chicago/Turabian StyleChen, Zixing, Wenmeng He, Thomas Chun Ning Leung, and Hau Yin Chung. 2021. "Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632" International Journal of Molecular Sciences 22, no. 13: 6782. https://doi.org/10.3390/ijms22136782