Anti cd16 cd32 blocking antibody

After 1 h of tumor digestion using DNAze I (Roche, Basel, Switzerland) and collagenase IV (Collagenase from Clostridium histolyticum; Sigma-Aldrich, Saint-Louis, MO, USA)—both 1 mg/mL) proceeded by mechanical tissue disintegration, the resultant cell suspension was filtered through a 100 µm cell strainer (Easystrainer, Greiner Bio-One, Kremsmünster, Austria). Subsequently, the cells were blocked using an anti-CD16/CD32 blocking antibody (BD Biosciences, San Jose, CA, USA). The cells were then incubated with anti-F4/80 MicroBeads (Miltenyi Biotec Inc., Auburn, CA, USA) and separated using magnetic columns (MACS® Cell Separation Columns—Miltenyi Biotec Inc., Auburn, CA, USA) following the manufacturer’s guidelines. The isolated cells were subsequently subjected to analysis for surface marker expression through flow cytometry or underwent short-term culture (72 h) to purify the material from debris. For the analysis of gene expression via qPCR, lipopolysaccharide (LPS; Sigma-Aldrich, Saint-Louis, MO, USA, 100 ng/mL) was added at a concentration of 100 ng/mL 24 h before lysis in TRI Reagent solution (Molecular Research Center, OH, USA). The cell culture supernatants were utilized for ELISA analysis.