Transduction to Label PDX Tumor Cells: Introducing Lentivirus Expressing Fluorescent Marker into the Tumor Cell In Vitro
Published: April 30, 2023
Abstract
Source: Hanna, C., et al. Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies. J. Vis. Exp. (2016).
The following video describes a technique of transduction to label (patient-derived xenografts) PDX tumor cells, which is used for introducing lentivirus expressing green-fluorescent protein and luciferase reporters into the tumor cell in vitro.
Protocol
1. Transduction of PDX-derived Tumor Cells Centrifuge dissociated tumor cells at 300 g x 5 min and resuspend in 2 ml mammosphere media. Count viable cells using trypan blue exclusion (Resuspend digested cells in 5 ml wash buffer and count both viable and non-viable cells using trypan blue exclusion. Briefly, mix 50 µl of cells and 50 µl of trypan blue and count trypan-blue excluding cells using a hemocytometer). Prepare 10 ml of mammosphere media containing 8 µg/ml polybrene (2 µ…
Representative Results
Figure 1: Tracking Transduction Efficiency in Dissociated PDX Cells. A) PDX-dissociated cells enriched for human epithelial cells (Lin+ depletion) 24 hr after transduction with pSIH1-H1-copGFP-T2A-puro lentiviral particles. B) PDX-dissociated cells from a separate experiment, without epithelial cell enrichment, 72 hr after transduction with pHAGE-EF1aL-luciferase-…
Disclosures
The authors have nothing to disclose.
Materials
| DMEM/F12 (1:1) | Hyclone | SH30023.01 | |
| bFGF | BD Biosciences | 354060 | |
| EGF | BD Biosciences | 354001 | |
| Heparin | Sigma | H4784 | |
| B27 | Gibco/Thermo Fisher | 17504-44 | |
| Anti-fungi-antibiotics | Hyclone | SV30010 | |
| HBSS Red Ca2+/Mg2+ free | Hyclone | SH30031.02 | |
| Hepes | |||
| Cultrex | Cultrex | 3433-005-01 | Basement Matrix Extract (BME) |
| 30 °C shaker | NewBrunswick Scientific CO. INC | Series 25 Incubator Shaker |
Cite This Article
Transduction to Label PDX Tumor Cells: Introducing Lentivirus Expressing Fluorescent Marker into the Tumor Cell In Vitro. J. Vis. Exp. (Pending Publication), e20214, doi: (2023).