Quanta qscript

Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) and cDNA was made using Quanta qScript (Quanta BioSciences, Gaithersburg, MD). The real time PCR was performed with Quanta Perfecta SYBR green fast mix and ABI prism 7500 sequence detection system (Applied Biosystems, Foster City, CA). Primers used were as follows:

FasL- FP 5′-TCTACCAGCCAGATGCACAC-3′, RP 5′-CAGAGGCATGGACCTTGAGT-3′

18s rRNA- FP 5′-CTCAACACGGGAAACCTCAC-3′, RP 5′-CGCTCCACCAACTAAGAACG-3′

The gene expression was analyzed by relative quantification using 2− ΔΔCt method by normalizing with 18s rRNA.

Differentiation into M1 and M2 macrophages was verified by qRT-PCR for inducible nitric oxide synthase (iNos) and arginase 1 (arg1), respectively. RNA was isolated with Qiagen RNeasy Midi Kit (Qiagen, Valencia, CA, USA) and Quanta qScript (Quanta Bioscience, Gaithersburg, MD, USA) was used for reverse transcription. qRT-PCR was performed using SYBR Green PCR Master Mix (Bio Rad, Hercules, CA, USA) with specific primer pairs (Supplementary Figure). Gene expression is presented relative to the housekeeping-gene guanine nucleotide-binding protein subunit beta-2-like 1 (GNB2L1). RNA samples, isolated from unpolarized or M1- or M2-polarized macrophages that had then been primed with LPS, were analyzed with the NanoString nCounter Analysis System (NanoString Technologies, Seattle, WA, USA). Unprimed BMDM served as control. Samples were prepared and data analyzed according to the manufacturer’s protocol. Gene expression is reported relative to the housekeeping-genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), hypoxanthinphosphoribosyltransferase 1 (HPRT1), and clathrin heavy chain 1 (CLTC).