Steady-state cytokine mRNA levels increased after CD15 cross-linking. Two micrograms of total RNA from unstimulated MM6 cells (as a control) or MM6 that were stimulated with anti-CD15 cross-linking (PM81, 10 μg/mL, 4 hours) were used as templates for cDNA synthesis. Aliquots were then amplified in the presence of 10-fold serial dilutions of the TNF-α competitors (617 bp) or IL-1β competitors (802 bp) (lanes 2 to 9). Lane 1 depicts control PCR whereby the reverse transcription reaction mixture was omitted. Lanes 2 to 5 represent the coamplification of cDNA from unstimulated MM6 cells and serial dilutions of the competitor DNA at 10−17, 10−18, 10−19, 10−20mol/L for TNF-α (A) and IL-1β (B), respectively. Lanes 6 to 9 represent the coamplification of cDNA from CD15-cross-linked (4 hours) and competitor serial dilutions of 10−17, 10−18, 10−19, 10−20 mol/L for TNF-α (A) and IL-1β (B), respectively. Aliquots of the final PCR products were resolved on a 1.6% EtBr-agarose gel.
