Cd3 apc h7 clone sk7

PBMCs were washed 3× with RPMI1640 culture medium (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) and resuspended in complete culture medium (CM: RPMI1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 50 µM 2-mercaptoethanol), at a concentration of 10⁶ cells/mL. PBMCs were cultured in triplicate in a 37 °C humidified chamber with 5% CO₂ for 12 h, in the presence or absence of mitogens (PI: 5 ng/mL phorbol myristate acetate and 1 μM ionomycin) (Sigma-Aldrich, Merck KGaA) or recombinant human leptin (R&D Systems) at a concentration of 200 or 500 or 800 ng/mL. At the end of culture, CD3+ T cells and CD14+ monocytes were isolated by cell sorting using a BD FACS Aria II flow cytometer (BD Biosciences, San Jose, CA, USA). The sorting strategy is shown in Figure 8. The enriched cell populations were used when purity was >95% for T cells and >90% for monocytes. The antibodies used for cell sorting and phenotyping were mouse monoclonal anti-human antibodies CD3-APC-H7 (clone SK7) and CD14-FITC (clone M5E2) (BD Biosciences). Fluorescence-minus-one (FMO) controls were used to identify any background spread of fluorochromes and to establish gating limits. Data were analyzed using BD FACS DIVA v.9 software (BD Biosciences).

Cells were stained with the following monoclonal antibodies; CD3- APC/H7 clone SK7 (BD Biosciences, San José, CA, US) and CD14-PeCy7 clone M5E2 (Nordic Biosite, Täby, Sweden). Whole blood and antibodies were incubated for 15 minutes at RT, thereafter erythrocytes were lysed with FACS Lysing Solution (BD Biosciences) for 15 minutes at room temperature (RT). The cells were permeabilized for 30 min at RT with Permeabilizing Solution 2 (BD Biosciences) and washed with Permeabilization buffer (Ebioscience, San Diego, CA, USA). Unspecific binding was blocked with 10% FCS and thereafter, cells were stained with antibodies against MMP-9 and TIMP-1, conjugated with FITC and PE respectively, (RnD Systems) for 30 min in 4°C. After washing in Permeabilization buffer followed by resuspension in PBS with 0.5% FCS, cells were analyzed using Beckman Coulter Gallios (Beckman Coulter, Miami Lakes, Florida, US). The obtained data was analyzed and visualized using the Kaluza 1.2 software (Beckman Coulter). When this analysis was performed, antibodies against TIMP-2 were not commercially available.