Cd138 bv421

The single cells were suspended in phosphate-buffered saline (PBS) with 0.1% bovine serum albumin as buffer. The human and mouse F_C receptors were blocked by human BD Fc block and mouse BD Fc block, respectively. The cell suspension was incubated with fluorochrome-conjugated monoclonal antibodies in dark, at room temperature, for 30 min. Cells were washed twice with buffer afterward, and then proceeded analysis on a Beckman Coulter Gallios flow cytometer. The dead cells were excluded by using 7-amino-actinonycin D (7-AAD). The data were analyzed on FlowJo software. The appropriate isotype antibodies were used as controls. The fluorochrome-conjugated monoclonal antibodies were used as follows; for human: CD138-BV421 (clone MI15, Biolegend), CD38-FITC (clone HB-7, BD), CD29-APC (clone TS2/16, Biolegend), CD49d-PE-CF594 (clone 9F10, BD), CD3-FITC (clone UCHT1, BD), CD19-BV421 (clone HIB19, BD), CD14-PE-CF594 (cloneMOP9, BD), CD45-BV510 (clone HI30, BD) and for mouse: CD138-BV421 (clone 281–2, BD), CD29-APC-eFluor780 (clone HMb1-1, eBioscience), CD49d-APC (clone R1-2, Biolegend), CD45-BV510 (clone 30-F11, Biolegend).

Cells were washed with PBS and then resuspended in binding buffer at a density of 1 × 10⁶ cells/ml. 100 μl of cell suspension was transferred to a 5 ml culture tube, next, 5μl CD138-BV421 and CD34-APC antibody (BD Bioscience, San Diego, CA, USA) were added to the tube. The mixture was vortexed and incubated for 15 min at 4°C in the dark. Finally, the samples were analysed by flow cytometry(BD Bioscience, San Diego, CA, USA).

Mice that were immunized as described above were sacrificed 4 weeks after the third immunization. Spleens were collected into RPMI containing 2% fetal bovine serum and manually homogenized using the back of a syringe plunger. Cells were filtered through 75 um mesh, washed 1x, and counted. 2x10⁷ splenocytes were stained for flow cytometry. All washes for the staining process were performed in PBS containing 2% fetal bovine serum and 2 mM EDTA. Cells were incubated with CD16/32 (Biolegend Cat# 101302) and 5.875 μg/ml of biotinylated Abp2D_RBD for 10 minutes, then washed 3x. A cocktail containing the following antibodies was prepared in BD Brilliant Staining Buffer (BD Cat. # 563794), all sourced from BioLegend unless otherwise indicated : Zombie NIR (Cat#423105), CD19-BV750 (Cat#115561), CD4-BV570 (Cat#100542), IgD-BV711 (Cat#405731), IgM-BV605 (Cat#406523), IgG1-BV510 (Cat#406621), Fas-PE (BD Cat# 554258), GL7-PcpCy5.5 (Cat# 144610), CD38-PE-Cy7 (Cat#102718), CD138-BV421 (Cat#142508), and streptavidin-APC-Fire-750 (Cat#405250). Invitrogen UltraComp eBeads were used for single colors. Flow cytometry data was collected using the Cytek Aurora with 4 laser 16V-14B-10YG-8R configuration and processed on FlowJo10 for Mac.