CTC Isolation from a Whole Blood Sample: A Method to Obtain CTCs from Murine Colorectal Cancer Model
Published: April 30, 2023
Abstract
Source: Kochall, S., et al. Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer. J. Vis. Exp. (2017).
This video describes a detailed protocol for isolating circulating tumor cells from the whole blood sample of a euthanized colorectal tumor mouse model. These isolated CTCs can be cultured and used for intended downstream analysis.
Protocol
All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Isolation of Circulating Tumor Cells from Whole Blood Samples NOTE: Obtain blood by transcutaneous cardiac puncture on anesthetized mice, followed by euthanasia. Ideally, 1,000 µL of blood are drawn into a syringe prefilled with 100 µL of an anticoagulant (Ethylenediaminetetraacetic ac…
Divulgations
The authors have nothing to disclose.
Materials
| Density gradient medium – Ficoll | StemCell – Lymphoprep | 7801 | |
| Alexa Fluor 488 anti-human CD326 (EpCAM) Antibody clone 9C4 | BioLegend | 324210 | |
| Alexa Fluor 488 anti-mouse CD326 (EpCAM) Antibody clone G8.8 | BioLegend | 118210 | |
| Petri Dish, ø 60 x 15 mm, 21 cm², Vent | Greiner bio-one | 628102 | |
| Fluorescence Cell Culture Microscope | Leica | ||
| Transferman 4r Micromanipulator | Eppendorf | ||
| CellTram Air | Eppendorf | aspiration pump connected to the micromanipulator | |
| Dmz Universal Microelectrode Puller | Dagan Corporation | required for the manufacturing of micro capillaries for single cell aspiration | |
| Prism Glass Capillaries | Dagan Corporation | ||
| PAP pen | Abcam | ||
| Dulbecco's Phosphate Buffered Saline | Life Technologies GmbH | 14190169 | picking buffer |
| Fetal Calf Serum (FCS) | BIOCHROM AG | S 0115 | picking buffer |
| Penicillin/Streptomycin (PenStrep) | Life Technologies GmbH | 15140122 | picking buffer |
| EDTA | Roth | 8040.1 | picking buffer |
| Immunohistochemistry | |||
| Purified anti-human CD326 (EpCAM) antibody clone 9C4 | BioLegend | 324201 | EpCAM immunohistochemistry |
| HRP rabbit anti-mouse IgG | Abcam | ab97046 | EpCAM immunohistochemistry |
Citer Cet Article
CTC Isolation from a Whole Blood Sample: A Method to Obtain CTCs from Murine Colorectal Cancer Model. J. Vis. Exp. (Pending Publication), e20342, doi: (2023).