Cell Cycle Analysis: An Approach to Study Cell Cycle Regulation of miRNA-transfected Lung Cancer Cells
Published: April 30, 2023
Abstract
Source: Hossian, A. K. M. N., et al. Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis. J. Vis. Exp. (2019).
miRNA or microRNA are key regulators of cell cycle progression. This video describes a flow cytometry-based cell cycle analysis of lung cancer cells transfected with miRNA, to study the effect of miRNA on cell cycle regulation. Inhibition of proliferation by halting cell cycle progression has shown potential to be a promising approach for cancer treatment.
Protocol
1. miR-143 and miR-506 transfection CAUTION: Use latex gloves, protective eyeglasses, and a laboratory coat while performing the described experiments. When required, use the biosafety cabinet with the cabinet fan on, without blocking the airways or disturbing the laminar airflow. Always set the protecting glass window to the appropriate height, as described by the manufacturer. Seed NSCLC A549 cells in a T25 cm2 flask/6/96 well plate in DMEM/F12K m…
Divulgations
The authors have nothing to disclose.
Materials
| DMEM 4.5 g/L Glucose, w/out Sodium Pyruvate, w/ L-Glutamine | VWR | VWRL0100-0500 | |
| Fetal Bovine Serum – Premium | Antlanta Biologicals | S11150 | |
| Fetal Bovine Serum (FBS) | Fisher Scientific | 10438026 | |
| Penicillin-streptomycin 10/10 | Antlanta Biologicals | B21210 | |
| Ribonuclease A from Bovine pancreas | Sigma | R6513-50MG | |
| Propidium Iodide | MP Biochemicals LLC | IC19545825 | |
| Ethanol, Absolute (200 Proof), Molecular Biology Grade | Fisher BioReagents | BP2818500 | |
| CO2 Incubator | Thermo Scientific | HERAcell 150i | |
| A549 Non Small Cell Lung Cancer Cells | ATCC | ATCC CCL-185 | |
| 96 well plate | CELLTREAT Scientific | 50-607-511 | |
| FACS Calibur Flowcytometer | Becton Dickinson | ||
| Thermo Scientific BioLite Cell Culture Treated Flasks | Thermo Scientific | 12-556-009 | |
| Temperature controlled centrifuge matchine | Thermo Scientific | ST16R | |
| Temperature controlled micro centrifuge matchine | Eppendrof | 5415R | |
| SpectraTube Centrifuge Tubes 15ml | VWR | 470224-998 | |
| Modfit LT | Verity Software | Step 2.15: Alternative software for analysis of cell cycle population distributions | |
| Fisherbrand Basix Microcentrifuge Tubes with Standard Snap Caps | Fisherbrand Basix | 02-682-002 | |
| hsa-miR-143-3p miRNA Mimic | ABM | MCH01315 | |
| hsa-miR-506-3p miRNA Mimic | ABM | MCH02824 | |
| F-12K Nutrient Mixture (Kaighn's Mod.) with L-glutamine | Corning | Corning 45000-354 | |
| Negative 80 °C Freezer | VWR | VWR40086A |
Citer Cet Article
Cell Cycle Analysis: An Approach to Study Cell Cycle Regulation of miRNA-transfected Lung Cancer Cells. J. Vis. Exp. (Pending Publication), e20238, doi: (2023).