A quantitative high-throughput chemotaxis assay using bioluminescent reporter cells.
Here we report on a novel biophotonic assay system for the detection and quantitation of chemotaxis, the directed movement of cells in response to chemokine concentration gradients. Our assay employs a firefly luciferase (ffLuc)-generated biophotonic signal to quantify cellular migration in 96-well microplate chemotaxis instruments. When compared to direct cell enumeration, the biophotonic reporter method is superior in accuracy, reproducibility, and sensitivity. As a proof-of-concept, we demonstrate the utility of this assay for quantifying the chemotactic response of ex vivo expanded ffLuc(+) primary human T-cells to recombinant human chemokines MCP-1, RANTES, and IP-10. The 96-well microplate format and in situ biophotonic detection of cells are amenable to high-throughput screening of peptides and small molecule libraries to identify agonists and antagonists of cellular chemotaxis, to analyze biological fluids for chemotactic activity, and to study chemotaxis in a variety of cell types.
Division of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
Reena P
RP
Vishwanath
Christine E
CE
Brown
Jamie R
JR
Wagner
Hunsar B
HB
Meechoovet
Araceli
A
Naranjo
Christine L
CL
Wright
Simon
S
Olivares
Dajun
D
Qian
Laurence J N
LJ
Cooper
Michael C
MC
Jensen
D000818:Animals; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D048888:Fireflies; D017930:Genes, Reporter; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics*; D013601:T-Lymphocytes / Q000502:physiology*
D018925:Chemokines; D008156:Luciferases