Exorneasy starter kit

We assessed whether exosomes contain mediators that modify S100A9 protein localization in MDSCs. Exosomes were purified from blood plasma or MDSC culture supernatants.in cell culture, exosomes were purified using exoEasy Maxi Kit per manufacturer's protocol (Qiagen, Valencia, CA). For RNA expression analysis, exosomes were purified and RNA was isolated using exoRNeasy Starter Kit (Qiagen).

Real-time qPCR (RT-qPCR) was used to determine the levels of Hotairm1 in exosomes, Gr1⁺CD11b⁺ cells, or S100A9 immunoprecipitates. Exosomes were purified from plasma or cell culture supernatants, and exosomal RNA was isolated using exoRNeasy Starter Kit (Qiagen). Total cellular RNA was isolated from Gr1⁺CD11b⁺ cells using TRIzol reagent (Invitrogen). Levels of Hotairm1 expression were measured using QuantiTect PCR Mastermix and RT2 lncRNAqPCR Assay primers (Qiagen). The expression level was calculated using the 2^−ΔΔCt cycle threshold method. Values were normalized to GAPDH RNA for total RNA or 18S RNA for exosomal RNA, amplified with QuantiTect Primer Assays. The PCR was performed in duplicate.