Bone marrow DC precursors were cultured with apoptotic neutrophils at the ratio of 1:1 (Ac:DC) at day0. Cells were harvested at day7 and washed with PBS. Then cells were spun down and put into TRIZOL (Invotrogen) for RNA extraction. cDNA was synthesized using High Capacity Reverse Transcription kits (Applied Biosystem). For real-time PCR, all Primer and probes mixes and Taqman fast master mixes were purchased from Applied Biosystems. Gene mRNA expression were quantified by using ABI 7500 fast real-time PCR system according to manufacturer’s instructions. The mRNA relative expression level of target gene was normalized to housekeeping gene 18s.
Taqman fast master mixes
Manufactured by Thermo Fisher Scientific
TaqMan Fast Master Mixes are optimized reagent mixes designed for fast and sensitive real-time PCR assays. They contain all the necessary components for efficient PCR amplification and detection, including a DNA polymerase, buffers, and fluorescent probes. These master mixes are suitable for a wide range of real-time PCR applications.
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2 protocols using taqman fast master mixes
1
Apoptotic Neutrophils Modulate Dendritic Cells
2
Isolation and Quantification of Immune Cells
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Cells from BAL, lung, and dMLNs were harvested as described above. CD11c+ cells were sorted by using CD11c MACs microbeads (Miltenyi Biotech). Sorted cells were spun down and put into 0.5 mL TRIzol (Invitrogen) for RNA extraction. cDNA was synthesized using High Capacity Reverse Transcription kit (Applied Biosystems, Foster City, CA). For real-time PCR, all primer and probe mixes and TaqMan fast master mixes were purchased from Applied Biosystems. For genes of interest, mRNA expression was quantified by using ABI 7500 fast real-time PCR system, according to the manufacturer's instructions. The mRNA relative expression level of target gene was normalized to housekeeping gene 18s.
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