In vitro evaluation of the effects of candidate immunosuppressive drugs: flow cytometry and quantitative real-time PCR as two independent and correlated read-outs

Journal of Immunological Methods
Mona G FloresDominic C Borie

Abstract

Immune monitoring may use flow cytometry or molecular biology techniques. Flow cytometry assays cells that are phenotypically characterized, whereas TaqMan RT-PCR starts with RNA extraction from unfractionated heterogeneous cell populations. We therefore wondered how the effects of immunosuppressive drugs on cytokine production in stimulated whole blood, as determined by flow cytometry, would correlate with those obtained with quantitative real-time PCR (TaqMan RT-PCR). Blood drawn from naive cynomolgus monkeys was exposed to incremental amounts of cyclosporine (CsA; 300, 600, 900 and 1200 ng/ml) or tacrolimus (TRL; 8, 20, 40 and 80 ng/ml) before lectin stimulation in vitro. Blood was then either stained for CD3, IFN-gamma, IL-2, IL-4, and TNF-alpha and analyzed on a flow cytometer with various gating strategies, or submitted to RNA extraction for analysis of the above mentioned cytokines mRNA transcripts using TaqMan RT-PCR. Both methods revealed a parallel dose-dependent inhibition of cytokine production in stimulated blood. The 50% inhibitory concentrations (IC(50)'s) ranged from 511-771 ng/ml (CsA) and 15-29 ng/ml (TRL) with flow cytometry, and from 275-529 ng/ml (CsA) and 11-48 ng/ml (TRL) with TaqMan RT-PCR for T-helper 1...Continue Reading

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Citations

Oct 10, 2008·Nephrology, Dialysis, Transplantation : Official Publication of the European Dialysis and Transplant Association - European Renal Association·Claudia SommererMartin Zeier
Dec 18, 2015·Journal of Veterinary Pharmacology and Therapeutics·C L FellmanA J Mackin
Nov 4, 2006·Transplant International : Official Journal of the European Society for Organ Transplantation·Geraldine RousvoalDominic C Borie
Apr 12, 2005·The Journal of Surgical Research·Mona G FloresDominic C Borie
May 26, 2005·International Immunopharmacology·Ming-Sing SiDominic C Borie
Jan 14, 2011·Veterinary Immunology and Immunopathology·C L FellmanA J Mackin
Aug 19, 2007·Journal of Leukocyte Biology·Georgina J ClarkDerek N J Hart
Feb 25, 2009·Immunopharmacology and Immunotoxicology·Brigitta RumbergerJohannes Donauer
Feb 10, 2007·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Matthias GrubeErnst Holler
Aug 26, 2019·Veterinary Immunology and Immunopathology·Claire L FellmanAndrew J Mackin

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