Lympholyte cell separation density gradient centrifugation media

All animal protocols have been approved by the Review Board and Institutional Ethics Committee of the Royan Institute (IR.ACERCR.Royan.REC.1395.59 21/06/2016). Mononuclear cells (MNCs) were obtained from rat spleens, followed by density separation using Lympholyte^® Cell Separation density gradient centrifugation media (Cedarlane, Canada). These MNCs were subjected to FACS positive selection of NK cells. Next, NK cells were isolated by positive selection of CD161+CD3- cells using FACS. Briefly, MNCs were stained using phycoerythrin (PE)-conjugated anti-CD161, and fluorescein isothiocyanate (FITC)-conjugated anti-CD3, according to the manufacturer’s protocol.
To obtain optimal NK cell expansion, we implemented two different protocols and compared their results. Isolated NK cells were cultivated in complete RPMI 1640 medium, with different enrichment approaches: the first group was supplemented with K562 as feeder cells and IL-2 (1000 IU/mL) and IL-15 (100 IU/mL), the second group was supplemented with cytokines alone. Both groups were incubated in a humidified incubator (5% CO2 at 37 °C).
In accordance with the previously published procedure, NK cells were activated by adding HSP70 (2 µg/mL) and IL-2 (100 IU/mL) to the culture medium, and further incubated under standard cell culture conditions for four days [29 (link)].

Visceral adipose tissue (VAT) was collected from OB-ND, OB-Dys and LC during surgery. In particular, omental fat was collected from obese patients and perirenal fat from LC. Blood was withdrawn before general anesthetic injection. VAT was transferred to the laboratory in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with GlutaMAX (ThermoFisher – Scientific), bovine serum albumin (BSA), penicillin/streptomycin (P/S), and HEPES, where it was processed within 90 min from the collection. VAT was finely minced and digested with collagenase IV (Sigma-Aldrich) resuspended in PBS with a final concentration of 2 mg/mL for 40 min at 37°. The sample was then washed with DMEM high glucose (10% FBS, 1% P/S, 1% Glutamine) and treated with Red Blood Lysis buffer (Biolegend) to obtain the stromal vascular fraction (SVF). After washing, the pellet of SVF was filtered and counted. Whole blood (WB) was collected simultaneously with VAT and used for flow cytometry analysis. Peripheral blood mononuclear cells (PBMCs) were obtained using Lympholyte Cell Separation density gradient centrifugation media (Cedarlane). After centrifugation, the PBMC ring was collected and washed two times with PBS.