Radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive T lymphocytes.
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.
Department of Neurosurgery, UCLA David Geffen School of Medicine.
Kate L
KL
Erickson
Department of Neurosurgery, UCLA David Geffen School of Medicine.
Michelle J
MJ
Hickey
Department of Molecular and Medical Pharmacology, UCLA David Geffen School of Medicine.
Yuki
Y
Kato
Department of Neurosurgery, UCLA David Geffen School of Medicine.
Colin C
CC
Malone
Department of Neurosurgery, UCLA David Geffen School of Medicine.
Geoffrey C
GC
Owens
Department of Neurosurgery, UCLA David Geffen School of Medicine; Department of Molecular and Medical Pharmacology, UCLA David Geffen School of Medicine.
Robert M
RM
Prins
Department of Neurosurgery, UCLA David Geffen School of Medicine; Brain Research Institute, UCLA David Geffen School of Medicine; Jonsson Comprehensive Cancer Center, UCLA David Geffen School of Medicine.
Linda M
LM
Liau
Department of Medicine, UCLA David Geffen School of Medicine; Jonsson Comprehensive Cancer Center, UCLA David Geffen School of Medicine.
Noriyuki
N
Kasahara
Department of Neurosurgery, UCLA David Geffen School of Medicine; Brain Research Institute, UCLA David Geffen School of Medicine; Jonsson Comprehensive Cancer Center, UCLA David Geffen School of Medicine; ckruse@mednet.ucla.edu.
Carol A
CA
Kruse
D000818:Animals; D001943:Breast Neoplasms / Q000276:immunology / Q000473:pathology; D002448:Cell Adhesion / Q000502:physiology; D002450:Cell Communication / Q000502:physiology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology*; D005260:Female; D005456:Fluorescent Dyes; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D008856:Microscopy, Fluorescence; D012190:Retroviridae / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000502:physiology*; D014161:Transduction, Genetic; D019076:Transgenes
D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins