Cd16 alexafluor700

Following thawing of cryopreserved PBMC and SFMC, cells were resuspended at a concentration of 1x10⁶ cells/100 μl in PBS with 10% heat-inactivated human AB serum (CTL-Europe GmbH, Bonn, Germany) to block surface Fc receptors. The cell suspension was incubated for 30 min at room temperature (RT) with the following mouse monoclonal anti-human antibodies: CD3 PerCP (Cat No 347344, BD Bioscience, Breda, The Netherlands), CD56 APC-H7 (Cat No 302216), CD16 AlexaFluor700 (Cat No 302026, BioLegend, San Diego, CA, USA), LLT1 APC (clone 402659, Cat No FAB3480A, R&D Systems, Abingdon, UK), CD14 eFluor 605NC (Cat No 93–0149, eBioscience, Vienna, Austria). Cells were analyzed using LSR II flow cytometer (BD Biosciences) and data analysis was performed with Kaluza analysis software (Beckman Coulter, Woerden, the Netherlands).

The following antibodies were used to label the CD markers: CD16-Alexa Fluor 700 (BioLegend, San Diego, CA, USA), CD63-peridinin chlorophyll protein (PerCP)-Cy5.5 (BioLegend), CD66-allophycocyanin (APC) (eBioscience, Santa Clara, CA, USA), CD14 PE-Cy7 (BioLegend), CD18-brilliant violet 421 (BV421) (BD), CD11b-APC-Cy7 (BioLegend) and CD64-phycoerythrin (PE) (BD). SONY SA3800 flow cytometer was used to acquire the data, and the channel voltages were calibrated manually with rainbow beads to normalize sample acquisition on different days. Compensation was performed with single-stained OneComp eBeads (eBioscience). Data were analysed by FlowJo software (v10; Tree Star), and the geometric mean fluorescence intensities (MFIs) were determined for each CD marker and H3Cit, as mentioned previously by Fine et al. [13 (link)]. Gating was then performed, as described by Fine et al. [13 (link)] (Figure A1), and the geometric mean fluorescence intensities were used for data analysis.