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

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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).

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