Mastercycler realpex 2

Tissues were collected at appropriate time points and snap-frozen in liquid nitrogen. Total RNA was isolated using a Qiagen RNeasy Plus Isolation Kit followed by cDNA synthesis (High Capacity cDNA RT kit, Applied Biosystems, 4374966). RT-PCR was performed using TaqMan Universal Master Mix II (Fisher, 4440038), according to manufacturer’s protocol. Primers used were: Myc (Fisher, Mm00487804_m1) and Tbp (Fisher, Mm00446973_m1). Samples were analysed in triplicate on an Eppendorf Mastercycler Realpex 2 with with Eppendorf Quantstudio design & Statistical Analysis Vl.2 Software. Tbp was used as an internal amplification control.

14 weeks post AdV-Cre KM mouse lungs were isolated 8 hours after IP injection with either oil or tamoxifen and then snap frozen in liquid nitrogen. Whole (tumor-laden) lung protein samples were isolated and incubated on a mouse inflammation antibody array (Abcam, ab133999) according to manufacturer instructions. The intensity of the signals was analyzed using ImageJ.
Total RNA (0.5-1 mg) isolated from F4/80^- lung- and tumor tissue or F4/80⁺ macrophages was reverse transcribed using the High Capacity cDNA RT kit (Applied Biosystems, 4374966). Real-time quantitative RT–PCR (Fast Sybr Green, Applied Biosystems, 4385612) was used to quantify mRNA levels. The TBP and β-actin genes were used as an internal amplification control. TBP forward primer: 5′-ACTTCGTGCAAGAAATGCTGAAT-3′, TBP reverse primer: 5′- CAGTTGTCCGTGGCTCTCTTATT-3′. β-actin forward primer: 5′-GACGATATCGCTGCGCTGG −3′, β-actin reverse primer: 5′-CCACGATGGAGGGGAATA-3′. MycER^T2 primers; forward: 5′-ATTTCTGAAGACTTGTTGCGGAAA-3′, reverse: 5′- GCTGTTCTTAGAGCGTTTGATCATGA-3′ (Murphy et al., Cancer Cell 14 (6), 2008). PD-L1 primers; forward: 5′-GACCAGCTTTTGAAGGGAAATG-3′, reverse: 5′-CTGGTTGATTTTGCGGTATGG-3′. VEGFA primers were from Bio-Rad (10025636, qMmuCED0040260). Real-time PCR reactions were performed on an Eppendorf Mastercycler Realpex 2 and analyzed with accompanying software.