Clone it2

In order to verify whether the differentiation of moDCs lead to a functional feasible DCs, we performed a response/maturation moDCs assay, based on the method described by Elkord et al. [2 (link)]. Herein, we used unstimulated DCs (immature DCs) as well as DCs stimulated with Lipopolysaccharide (LPS) from Escherichia coli (026: B6) at 0.5 μg/ml (Sigma-Aldrich) for 24 hours. After LPS stimulation the cells cultures were incubated at 37°C and 5% CO₂ for 24 hours.
Subsequently, cells were removed from plates, washed in PBS pH 7.4, transferred into tubes (Falcon Inc., USA) and stained for 20 min in the dark at 4°C with anti-CD86 APC (Dilution 1:100; Clone IT2.2; BioLegend, USA; cat 305412) and anti-CD80 APC Alexa 750 (Dilution 1:40; Clone HA5.2B7; Beckman Coulter, USA; cat PNB30643). Anti-CD14 PE-Dazzele 594 and anti-CD209 PE were used for phenotyping monocytes and moDCs respectively. For staining and analysis procedures, we used the same protocol as described in section 2.5. Regarding fluorescence detection, the following lasers and filters were used: At 488 laser FACSAria 576/26 filter was used for PE detection, FACSAria 610/20 filter was used for PE-Dazzle594 detection. At 635 laser FACSAria 660/20 filter was used for APC detection and FACSAria 780/60 filter was used for APC- AlexaFluor 750 detection.

MDMs were detached by incubation with TrypLE™ Express solution (Life Technologies) at 37°C for 10 min. For the analysis of surface markers, monocytes and MDMs were incubated for 20 min with saturating concentrations of monoclonal antibodies against HLA-DR (BioLegend; clone L243), CD86 (BioLegend; clone IT2.2), CD206 (BioLegend; clone 15-2), and CD163 (BioLegend; clone GHI/61). Cells were also stained with dihydrorhodamine 123 and 4-amino-5-methylamino-2',7′-difluorofluorescein diacetate, all from Invitrogen.
For experiments with neutrophils, cells were stained with BD Horizon^TM Fixable Viability Stain 450 (BD Biosciences), followed by the surface monoclonal mouse antihuman-conjugated antibodies: anti-CD15-PE (clone H198) and anti-CD11b-FITC (ICRF44) from BioLegend. Intracellular staining was performed after fixation and permeabilization with Fix/Perm kit (eBiosciences), with the antibodies anti-IL-1β-FITC (clone JK1B-1), anti-IL-6-APC (MQ2-13A5), anti-TNFα-APC (Mab11) from BioLegend, and anti-IL-10-FITC (BT-10) from eBioscience.