Activation of Cytokine and Membrane Protein Expression

Leucocyte stimulation was required for intracellular cytokine and Pgp1 expression [11 (link),13 ]. Consistent with previous reports [14 (link),15 (link)] preliminary experiments showed GCR expression was detected in less than 2% of unstimulated T cells but GCR expression was significantly upregulated in T and NKT-like cells following the same stimulation as for intracellular cytokine and Pgp1 detection. One mL aliquots of blood diluted 1:2 with RPMI 1640 medium (Gibco, New York, USA) supplemented with 125 U/mL penicillin and 125 U/mL streptomycin) were placed in a 10 mL sterile conical PVC tubes (Johns Professional Products, Sydney, Australia). Phorbol myristate (25 ng/mL) (Sigma, Sydney, Australia) and ionomycin (1 μg/mL) (Sigma) was added. Brefeldin A (10 μg/mL) (Sigma) was added as a “Golgi block” and the tubes re-incubated in a humidified 5% CO₂/95% air atmosphere at 37°C for 16 h. Preliminary studies showed that addition of Brefeldin A was shown not to affect Pgp1 or GCR expression in T and NKT-like cells (not shown).