This study aims to assess the impact of FcR blocking reagents on detection of BCR IgH isotypes IgM, IgD, IgA1-2, and IgG1-4. Blood samples were collected from healthy volunteers in lithium heparin tubes, followed by the isolation of PBMCs utilizing BioColl density gradient centrifugation. The blood samples were diluted at a ratio of 1:1 in PBS with a pH of 7.4 and then centrifuged at 800 g for 20 minutes at RT. The PBMC interphases were harvested and washed three times with PBS containing 2 mM EDTA. Subsequently, PBMCs were resuspended in cRPMI medium. PBMCs were treated with five different FcR blocking reagents prior to staining. For each donor, PBMCs were divided into two groups, with each group containing five samples. One group was stained directly with an immunophenotyping panel without being washed following the application of FCR blocking reagent. The other group was applied washing before staining.Ultimately, the samples were incubated with an 8-color immunophenotyping panel targeting B-cell markers: Target Fluorochrome Producer Clone Isotype Dilution CD19 APC-eFluor780 eBioscience HIB19 Mouse, IgG1, kappa 1:100 IgM PerCP/Cy5.5 Biolegend MHM-88 Mouse, IgG1, kappa 1:100 IgD AF700 Biolegend IA6-2 Mouse IgG2a, kappa 1:100 IgA VioBlue Miltenyi IS11-8E10 Mouse, IgG1, kappa 1:200 IgA2 PE Miltenyi IS11-21E11 Mouse, IgG1, kappa 1:100 IgG1 AF488 AF488/Lumiprobe MH161-1 Mouse, IgG2b, kappa 1:600 IgG1 Dylight550 Dylight/Innova Bio MH161-1 Mouse, IgG2b, kappa 1:500 IgG2 Dylight550 Dylight/Innova Bio HP6002 Mouse, IgG1, kappa 1:100 IgG3 AF488 AF488/Lumiprobe MH163-1 (HP6095) Mouse, IgG2b, kappa 1:400 IgG4 APC Cytognos SAG4 Mouse, IgG1, kappa 1:400 CD3 BV605 Biolegend OKT3 Mouse IgG2a, kappa 1:200 CD14 BV605 Biolegend 63D3 Mouse, IgG1, kappa 1:200 CD16 BV605 Biolegend 3G8 Mouse, IgG1, kappa 1:200 Viability dye Zombie yellow Biolegend 1:100 Cells were analyzed on a FACS Aria IIIu (Becton, Dickinson and Company, Franklin Lakes, NJ). B cells were gated as live ZombieYellow-CD3-CD14-CD16-CD19+ lymphocyte. IgM and IgD expression was used to differentiate class-switched (IgM-IgD-) and non-switched (IgM+IgD+) B cells. Next, the IgA+ population, gated from switched B cells, was segregated into IgA1+ (gated as IgA+IgA2-) and IgA2+ (IgA+IgA2+) B cells. The IgA- population was segregated into IgG1+, IgG2+, IgG3+ and IgG4+ B cells. Gates were set based on fluorescence minus one (FMO) controls.To evaluate the effectiveness of the different blocking reagents used in our study on preventing non-specific binding of mouse monoclonal antibodies (mAbs), PBMCs were incubated with isotype control antibodies. Mouse IgG1, IgG2a, and IgG2b antibodies, all conjugated with PE, were evaluated for their non-specific binding to CD14+ monocytes. Prior to staining, the cells were divided into two groups and were treated with FcR blocking reagent as mentioned above. The details concerning the reagents are below: Target Fluorochrome Brand Clone Isotype Dilution IgG1 PE Biolegend MOPC-21 Mouse, IgG1, kappa 1:100 IgG2a PE Biolegend MOPC-173 Mouse IgG2a, kappa 1:100 IgGb PE Biolegend MPC-11 Mouse, IgG2b, kappa 1:100 CD14 BV605 Biolegend 63D3 Mouse, IgG1, kappa 1:200 Viability dye Zombie green Biolegend 1:100 The analysis of all flow cytometry data was performed using FlowJo v10 software (BD Biosciences). Statistical analysis was conducted using GraphPad Prism 9 software (GraphPad, La Jolla, USA).