| Chapter title |
Cell Cycle Synchronization of HeLa Cells to Assay EGFR Pathway Activation
|
|---|---|
| Chapter number | 13 |
| Book title |
ErbB Receptor Signaling
|
| Published in |
Methods in molecular biology, January 2017
|
| DOI | 10.1007/978-1-4939-7219-7_13 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-7218-0, 978-1-4939-7219-7
|
| Authors |
Ping Wee, Zhixiang Wang, Wee, Ping, Wang, Zhixiang |
| Abstract |
Progression through the cell cycle causes changes in the cell's signaling pathways that can alter EGFR signal transduction. Here, we describe drug-derived protocols to synchronize HeLa cells in various phases of the cell cycle, including G1 phase, S phase, G2 phase, and mitosis, specifically in the mitotic stages of prometaphase, metaphase, and anaphase/telophase. The synchronization procedures are designed to allow synchronized cells to be treated for EGF and collected for the purpose of Western blotting for EGFR signal transduction components.S phase synchronization is performed by thymidine block, G2 phase with roscovitine, prometaphase with nocodazole, metaphase with MG132, and anaphase/telophase with blebbistatin. G1 phase synchronization is performed by culturing synchronized mitotic cells obtained by mitotic shake-off. We also provide methods to validate the synchronization methods. For validation by Western blotting, we provide the temporal expression of various cell cycle markers that are used to check the quality of the synchronization. For validation of mitotic synchronization by microscopy, we provide a guide that describes the physical properties of each mitotic stage, using their cellular morphology and DNA appearance. For validation by flow cytometry, we describe the use of imaging flow cytometry to distinguish between the phases of the cell cycle, including between each stage of mitosis. |
X Demographics
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Japan | 1 | 50% |
| Unknown | 1 | 50% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 2 | 100% |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 31 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Bachelor | 8 | 26% |
| Student > Master | 7 | 23% |
| Student > Ph. D. Student | 3 | 10% |
| Researcher | 2 | 6% |
| Lecturer | 1 | 3% |
| Other | 0 | 0% |
| Unknown | 10 | 32% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 11 | 35% |
| Chemistry | 2 | 6% |
| Agricultural and Biological Sciences | 1 | 3% |
| Immunology and Microbiology | 1 | 3% |
| Pharmacology, Toxicology and Pharmaceutical Science | 1 | 3% |
| Other | 4 | 13% |
| Unknown | 11 | 35% |