Radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive T lymphocytes

Kate L. Erickson, Michelle J. Hickey, Yuki Kato, Colin C. Malone, Geoffrey C. Owens, Robert M. Prins, Linda M. Liau, Noriyuki Kasahara, Carol A. Kruse

Research output: Contribution to journalArticle

Abstract

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number96
DOIs
StatePublished - Feb 11 2015
Externally publishedYes

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T-cells
Tumors
Assays
Extracellular Matrix Proteins
Cells
T-Lymphocytes
Monolayers
Fluorescence
Proteins
Imaging techniques
Fluorescence microscopy
Stainless Steel
Polytetrafluoroethylene
Cytotoxicity
Fluorescent Dyes
Polytetrafluoroethylenes
Agar
Labels
Deposits
Stainless steel

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive T lymphocytes. / Erickson, Kate L.; Hickey, Michelle J.; Kato, Yuki; Malone, Colin C.; Owens, Geoffrey C.; Prins, Robert M.; Liau, Linda M.; Kasahara, Noriyuki; Kruse, Carol A.

In: Journal of visualized experiments : JoVE, No. 96, 11.02.2015.

Research output: Contribution to journalArticle

Erickson, Kate L. ; Hickey, Michelle J. ; Kato, Yuki ; Malone, Colin C. ; Owens, Geoffrey C. ; Prins, Robert M. ; Liau, Linda M. ; Kasahara, Noriyuki ; Kruse, Carol A. / Radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive T lymphocytes. In: Journal of visualized experiments : JoVE. 2015 ; No. 96.
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