Ficoll hypaque gradients

The study was approved by the Ethics Committee of IRCCS Neuromed (No. 20160106–1006) and conducted in accordance with the Declaration of Helsinki. All participants gave written informed consent. Peripheral blood mononuclear cells from diabetic patients were isolated over Ficoll–Hypaque gradients (lymphocyte separation medium; MP Biomedicals, Aurora, OH,
USA).

Healthy peripheral blood mononuclear cells were separated over Ficoll-Hypaque gradients (MP Biomedicals, Aurora, OH, USA). PBMC were grown in RPMI 1640 (Invitrogen, San Diego, CA, USA), supplemented with 2 mM l-glutamine, 50 ng/mL, streptomycin, 50 units/mL penicillin, and 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT, USA). All volunteers provided written informed consent in agreement with the Declaration of Helsinki to the use of their residual buffy coats for research aims with approval from the University Hospital of Salerno Review Board.
Human immortalized keratinocytes (HaCaT) were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, Grand Island, NY, USA) supplemented with 2 mM l-glutamine, 50 ng/mL, streptomycin, 50 units/mL penicillin, and 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT, USA). HaCaT cells were kindly provided by Giuseppe Monfrecola (Department of Experimental Dermatology, University of Naples, Naples, Italy).