Dataset Information

Antibody/Ligand-Target Receptor Internalization Assay Protocol Using Fresh Human or Murine Tumor Ex Vivo Samples.

ABSTRACT: We describe an ex vivo EGF ligand internalization assay using fresh patient tumor biopsies to determine how antigen targets will be trafficked before patients receive mAb treatment. This protocol facilitates a sensitive and reproducible indication as to mAbs surface retention times during treatment. EGF uptake protocols can also be used to analyze EGFR heterogeneity and localization of EGFR in both tumor and xenograft tissue. The technology can be adapted to analyze other receptors such as PD-L1 for which methods are provided. For complete details on the use and execution of this protocol, please refer to Joseph et al. (2019) and Chew et al. (2020).

SUBMITTER: Joseph SR

PROVIDER: S-EPMC7580087 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Antibody/Ligand-Target Receptor Internalization Assay Protocol Using Fresh Human or Murine Tumor <i>Ex Vivo</i> Samples.

Joseph Shannon R SR Lum Benedict B Banushi Blerida B Barry Rachael R Panizza Benedict B Walpole Euan E Simpson Fiona F

STAR protocols 20200813 2

We describe an <i>ex vivo</i> EGF ligand internalization assay using fresh patient tumor biopsies to determine how antigen targets will be trafficked before patients receive mAb treatment. This protocol facilitates a sensitive and reproducible indication as to mAbs surface retention times during treatment. EGF uptake protocols can also be used to analyze EGFR heterogeneity and localization of EGFR in both tumor and xenograft tissue. The technology can be adapted to analyze other receptors such a ...[more]

PMID: 33111120

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Project description:Borrelia burgdorferi, the causative agent of Lyme disease, has been recently shown to form biofilm structures in vitro and in vivo. Biofilms are tightly clustered microbes characterized as resistant aggregations that allow bacteria to withstand harsh environmental conditions, including the administration of antibiotics. Novel antibiotic combinations have recently been identified for B. burgdorferi in vitro, however, due to prohibiting costs, those agents have not been tested in an environment that can mimic the host tissue. Therefore, researchers cannot evaluate their true effectiveness against B. burgdorferi, especially its biofilm form. A skin ex vivo model system could be ideal for these types of experiments due to its cost effectiveness, reproducibility, and ability to investigate host-microbial interactions. Therefore, the main goal of this study was the establishment of a novel ex vivo murine skin biopsy model for B. burgdorferi biofilm research. Murine skin biopsies were inoculated with B. burgdorferi at various concentrations and cultured in different culture media. Two weeks post-infection, murine skin biopsies were analyzed utilizing immunohistochemical (IHC), reverse transcription PCR (RT-PCR), and various microscopy methods to determine B. burgdorferi presence and forms adopted as well as whether it remained live in the skin tissue explants. Our results showed that murine skin biopsies inoculated with 1 × 107 cells of B. burgdorferi and cultured in BSK-H + 6% rabbit serum media for two weeks yielded not just significant amounts of live B. burgdorferi spirochetes but biofilm forms as well. IHC combined with confocal and atomic force microscopy techniques identified specific biofilm markers and spatial distribution of B. burgdorferi aggregates in the infected skin tissues, confirming that they are indeed biofilms. In the future, this ex vivo skin model can be used to study development and antibiotic susceptibility of B. burgdorferi biofilms in efforts to treat Lyme disease effectively.

Dataset Information

Antibody/Ligand-Target Receptor Internalization Assay Protocol Using Fresh Human or Murine Tumor Ex Vivo Samples.

Publications

Antibody/Ligand-Target Receptor Internalization Assay Protocol Using Fresh Human or Murine Tumor <i>Ex Vivo</i> Samples.

Similar Datasets

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