Mesothelial Clearance Assay: An In Vitro Technique to Quantify the Invasion Ability of Ovarian Cancer Spheroids into Mesothelial Cell Monolayers
Published: April 30, 2023
Abstract
Source: Davidowitz, R. A. et al. In Vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis. J. Vis. Exp. (2012)
This video describes a technique to quantitatively measure the interaction of ovarian cancer spheroids with mesothelial cells, using fluorescently labeled cells and time-lapse video microscopy. This in vitro assay models the interaction between metastasizing ovarian cancer cell spheroids and mesothelial cells lining the peritoneal cavity in vivo.
Protocol
1. Ovarian Cancer Cell Spheroid Formation RFP-expressing ovarian cancer cells are cultured in 10% Base Medium (a custom cell culture medium containing a 50:50 mixture of 199 and MCDB105, 10% inactivated fetal bovine serum, and 1% pen-strep). To express RFP in unlabeled ovarian cancer cells, transfect the cells with a plasmid containing RFP and select for cells expressing RFP. Alternatively, viral vectors can be used to express fluorescent proteins transiently, or cells can be pre-incu…
Açıklamalar
The authors have nothing to disclose.
Materials
| OVCA433 Ovarian Cancer Cells | Gift from Dr. Dennis Slamon | ||
| ZT Mesothelial Cells | Gift from Dr. Tan Ince | ||
| Medium 199 | GIBCO by Life Technologies | 19950 | |
| MCDB105 | Cell Applications Inc. | 117-500 | |
| FBS-heat inactivated | GIBCO, by Life Technologies | 10082 | |
| Pen-Strep | GIBCO, by Life Technologies | 15070 | |
| 96 well plates | Corning | 3799 | |
| Polyhydroxyethylmethacrylate (poly-HEMA) 192066-25G | Sigma-Aldrich | For poly-HEMA solution dissolve 6mg poly-HEMA powder in 1ml of 95% EtOH | |
| EtOH | Pharmco-AAPER | 111ACS200 | Dilute to 95% in dH20 |
| Cell culture hood | Nuaire | NU-425-300 | |
| Tissue culture incubator | Thermo Fisher Scientific, Inc. | 3110 | |
| Incubator for poly-HEMA plates 305 | Labline Instruments | Imperial III | |
| Tabletop centrifuge | Heraeus Instruments | 75003429/01 | |
| 6 well glass-bottom dish | MatTek Corp. | P06G-1.5-20-F | |
| Fibronectin | Sigma-Aldrich | F1141-1MG | |
| PBS | Cellgro | 21-040-CV | |
| Microscope | Nikon Instruments | Ti-E Inverted Motorized Fluorescence time-lapse microscope with integrated Perfect Focus System | |
| Lens | Nikon Instruments | 20X-0.75 numerical apeture | |
| Halogen transilluminator | Nikon Instruments | 0.52 NA long working distance condenser | |
| Excitation and emission filters | Chroma Technology Corp. | GFP Ex 480/40, Em 525/50 RFPmCherry Ex 575/50 Em 640/50 | |
| Transmitted and Epifluoresce light path | Sutter Instrument Co. | Smart Shutters | |
| Linear-encoded motorized stage | Nikon Instruments | ||
| Cooled charged-coupled device camera | Hamamatsu Corp. | ORCA-AG | |
| Microscope incubation chamber with temperature and CO2 control | Custom Made | ||
| Vibration isolation table | TMC | ||
| NIS-Elements software | Nikon Instruments | Version 3 |
Etiketler
Bu Makaleden Alıntı Yapın
Mesothelial Clearance Assay: An In Vitro Technique to Quantify the Invasion Ability of Ovarian Cancer Spheroids into Mesothelial Cell Monolayers. J. Vis. Exp. (Pending Publication), e20382, doi: (2023).