RNA isolation from cells treated for 24 h was done using TRIZOL.32 (link) The RNA was stored at −80 °C after
quantification using a multimode microplate reader (BioTek Synergy
H1) until further use. cDNA synthesis was done using the iScript cDNA
synthesis kit (BioRad) according to the manual provided by the manufacturer.
Reaction parameters: priming for 5 min at 25 °C, cDNA synthesis
for 20 min at 46 °C, reverse transcriptase inactivation at 95
°C for 1 min, and hold at 4 °C for infinity. Quantitative
RT-PCR was performed after mixing the SYBR green PCR master mix (BioRad)
with cDNA and gene-specific primers for NF-κB, RAGE, iNOS, MHC-II,
CD86, CD206, ARG1, and GAPDH (Supporting Table 1 ) and running the reaction in CFX 96 RT-PCR systems (BioRad,
California).33 (link) The results were expressed
as a fold increase relative to GAPDH.
quantification using a multimode microplate reader (BioTek Synergy
H1) until further use. cDNA synthesis was done using the iScript cDNA
synthesis kit (BioRad) according to the manual provided by the manufacturer.
Reaction parameters: priming for 5 min at 25 °C, cDNA synthesis
for 20 min at 46 °C, reverse transcriptase inactivation at 95
°C for 1 min, and hold at 4 °C for infinity. Quantitative
RT-PCR was performed after mixing the SYBR green PCR master mix (BioRad)
with cDNA and gene-specific primers for NF-κB, RAGE, iNOS, MHC-II,
CD86, CD206, ARG1, and GAPDH (
California).33 (link) The results were expressed
as a fold increase relative to GAPDH.
