DOI: 10.5281/zenodo.13326705
title: Strategies for Flow Cytometric Profiling of BCR Immunoglobulin Heavy Chain Isotypes: A Comparative Assessment of Fc Receptor Blocking Agents

description: 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).