Veterinary immunology and immunopathology vol:81 issue:1-2 pages:97-112
To analyze the correlation between cytokine mRNA transcription and secretion, interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) were quantified by enzyme-linked immunosorbent assay and IL-2 by bioassay and compared with their mRNA levels, determined by reverse transcription polymerase chain reaction (RT-PCR). For this purpose, peripheral blood monomorphonuclear cells (PBMC) were stimulated in vitro with the lectins pokeweed mitogen (PWM) phytohemagglutinin (PHA) and concanavalin A (ConA), respectively, and with F4 fimbriae in an antigen-specific assay. Analyses were performed 4, 8, 16, 24 and 48 h after stimulation on the stimulated PBMC for mRNA and on the respective culture supernatants for proteins. RT-PCR products were quantified by densitometric scanning of the electrophoresis bands and related to the band intensity of the housekeeping gene, cyclophilin.