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

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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).

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