Nanomaterials trigger functional responses in primary human immune cells
Nanomaterials trigger functional responses in primary human immune cells
Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation (1,200g, 20mins) of buffy coats from healthy volunteer blood donors. Following erythrolysis (MiltenyiBiotec #130-094-183), NK cells and T cells were isolated from PBMC by negative magnetic cell-sorting (MiltenyiBiotec #130-092-657and #130-096-535) following manufacturer instructions. The cell purity was then controlled by flow cytometry on a Attune NxT (Invitrogen) flow cytometer using PE anti-CD56 (1:100, MiltenyiBiotec # 130-113-312) and FITC anti-CD3 (1:100, MiltenyiBiotec #130-126-882) staining and ranged from 90% to 99.2% (median 96.3%). NK- and T cells (106 cells/mL) were cultured for further experiment in R10 medium:  RPMI 1640 (Gibco #72400054) containing 2mM Glutamine, 25mM HEPES and completed with 10 % v/v of (fetal bovine serum) FBS (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin (PanBiotech #P06-07100) and 50 μM beta-mercaptoethanol (Gibco #11508916).  After 48h of culture, NK and T cells untreated or treated with NPs at the indicated concentrations were harvested and washed in ice-cold PBS. Total RNA was isolated using the RNAeasy kit (Qiagen #74136) with on-column DNAse I digestion (Qiagen #79256) according to manufacturer's instruction. RNA was eluted in 50μL final volume, and its concentration was assessed with the NanoPhotometer® N60 (Implemen). RNA integrity was assessed by Bioanalyzer (total RNA Pico Kit, 2100 Instrument, Agilent Technologies, Paolo Alto, CA, USA). All samples had RNA integrity numbers above 7. Sequencing libraries were prepared using “NEBNext Ultra II Directional RNA Library Prep Kit for Illumina“ and enriched in mRNA using “NEB Ultra II polyA m RNA magnetic isolation” kit(New England Biolabs, Ipswich, MA, USA).