Anti human cd3 alexa fluor 700 okt3

We analyzed the intensity of α4-integrin expression on B-cell subsets using blood samples of HCs. Human peripheral blood mononuclear cells (PBMCs) of 8 HCs (mean age 30.5 years, male:female 5:3) were isolated using Pancoll solution (PAN-Biotech Pancoll human, density: 1.077 g/mL) and stored in liquid nitrogen until further use. After thawing, cells were incubated with the FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained using anti-human CD3-Alexa Fluor 700 (OKT3; eBioscience, San Diego, CA), CD14-Alexa Fluor 700 (M5E2, BioLegend, San Diego, CA), CD20-PerCP (2H7 BioLegend), IgD-PerCP (IA6-2, BioLegend), CD19-APC/Fire 750 (HIB19, BioLegend), CD27-Brilliant Violet 605 (O323; BioLegend), CD38-eFluor 450 (HB7; eBioscience), α4-integrin—(CD49d) FITC (9F10, BioLegend), and FITC Mouse IgG1, κ Isotype Control (MOPC-21, BioLegend). B-Cell subpopulations were analyzed within the CD3⁻CD14⁻, CD19⁺ population. In this article, naive B cells were defined as CD38⁻ CD27⁻; memory B cells were defined as CD38⁻CD27⁺ and plasmablasts as CD38⁺CD27⁺. Expression levels of α4-integrin were analyzed as delta-mean fluorescence intensity (ΔMFI) (MFI of VLA-4-FITC-staining − MFI of fluorescence minus one control without α4-integrin—FITC). Flow cytometry (FACS) data were analyzed using FlowJo 10.6.1 software (Ashland, OR).

Cells were stained using anti-human CD3-Alexa Fluor 700 (OKT3; eBioscience), CD19-APC/Fire 750 (HIB19; BioLegend, San Diego, CA), CD27-Brilliant Violet 605 (O323; BioLegend), CD38-eFluor 450 (HB7; eBioscience), CD138-PE (Mi15; STEMCELL Technologies, Vancouver, Canada), FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany), and TO-PRO-3 (Invitrogen, Eugene, OR).