Strategies for Flow Cytometric Profiling of BCR Immunoglobulin Heavy Chain Isotypes: A Comparative Assessment of Fc Receptor Blocking Agents

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).