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Blood, Vol. 105, Issue 3, 1319-1328, February 1, 2005
Minute numbers of contaminant CD8+ T cells or CD11b+CD11c+ NK cells are the source of IFN- in IL-12/IL-18-stimulated mouse macrophage populations
Blood Schleicher et al.
105: 1319
Supplemental materials for: Schleicher et al, Vol 105, Issue 3, 1319-1328
Supplemental Materials and Methods
Conventional RT-PCR and real-time RT-PCR
For conventional RT-PCR, the PCR reactions contained 1 µL cDNA, 1 × PCR buffer (Invitrogen), 2mM MgCl2, 250µM dNTP each, 1 unit of recombinant Taq Polymerase (Invitrogen), and 0.2µM primers (Thermo Electron, Ulm, Germany) and were subjected to the following amplification scheme: 5 minutes at 95°C, 40 cycles of 30 seconds at 95°C, 30 seconds at 62°C (IFN )/58°C (HPRT), and 30 seconds at 72°C. PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and visualized by ultraviolet fluorescence. Primers for mouse IFN were 5´-GGTGACATGAAAATCCTGCAGAGCCAGA-3´ (sense) and 5´-GAATGCTTGGCGCTGGACCTG TGGGTTG-3´ (antisense). Primers for the murine housekeeping gene hypoxanthine-guanine-phosphoribosyltransferase (HPRT) were 5´-TGATCAGTCAACGGGGGACA-3´ (sense) and 5´-TTCGAGAGGTCCTTTTCACCA-3´ (antisense). Both primer pairs failed to amplify a product from genomic mouse DNA.
For quantitative real-time PCR, the reaction mixture (20 µL) was prepared with the 2 × Taqman Universal Mastermix (Applied Biosystems) and contained 100 ng cDNA (9 µL) and 1µL Assays-on-Demand (Applied Biosystems) including forward and reverse primers and the 6-carboxy-fluorescein (FAM)–labeled probe for the target gene (assay for mIFN was no. Mm00801778_m1; for mouse HPRT, it was Mm00446968_m1). PCR amplification and detection were performed with an ABI Prism 7900 sequence detector (Applied Biosystems) using the following profile: 2 minutes at 50°C (AmpErase UNG deactivation), 10 minutes at 95°C (AmpliTaq Gold activation) and 40 cycles of 15 seconds at 95°C (denaturation) and 60 seconds at 60°C (annealing, extension). Each sample was amplified in triplicate. mRNA levels were calculated with the sequence detector SDS2.1 software (Applied Biosystems), using the comparative cycle threshold method1 and normalized to the endogenous control HPRT. The threshold cycle (Ct) is defined as the fractional cycle number at which the reporter fluorescence generated by the cleavage of the FAM-labeled probe passes a fixed threshold above baseline.1 The mRNA levels are reported as the fold difference relative to the calibrator cDNA (derived from purified C57BL/6 BMM cultured for 24 hours without further stimulation; Figure 7B). In cases in which the Ct number for unstimulated macrophages was greater than or equal to 40, the absolute Ct values are reported (Table S1).
Reference
1. Fehninger TA, Shah MH, Turner MJ, et al. Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response. J Immunol. 1999;162:4511-4520.
Files in this Data Supplement:
- Table S1 -
Ct values of real-time PCR analyses of IFN and HPRT mRNA expression in PECs and BMMs of Rag2−/−c−/− mice (PDF, 82KB): PECs and BMMs of Rag2−/−c−/− mice were stimulated with 1 ng/mL IL-12 with or without 10 ng/mL IL-18; 10 ng/mL IL-18; or 1 µg/mL LPS for 24 hours. RNA was prepared, reverse-transcribed, and analysed by real-time PCR for HPRT and IFNmRNA expression. Each cDNA sample was analyzed in triplicate. The numbers given are means of the Ct (threshold cycle) values ± SD. For a detailed description of the threshold cycle (Ct) parameter, see "Material and methods" and Fehninger et al.1
- Figure S1 -
IFN mRNA expression in RAW264.7 macrophages (PDF, 36 KB): RAW264.7 macrophages were incubated with 1 ng/mL IL-12 with or without 10 ng/mL IL-18; 10 ng/mL IL-18; or LPS (1 µg/mL) for 24 hours. RNA was prepared and analysed by conventional RT-PCR for HPRT and IFN. Results shown are from one of 2 similar experiments.
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