Four weeks after finishing therapy mice were euthanized and bone marrow was immediately harvested. Single cell suspensions were made by successive passes through a fine gauge needle. Red blood cells were lysed by suspension in ACK lysing buffer (Lonza). Cells were incubated with anti-CD16/CD32 (FcBlock, BD Biosciences) before incubation with anti-CD138-PE (BioLegend), -CD38-FITC (BioLegend), -CD19-BV421 (BioLegend), and -B220/CD45R-APC (BD Biosciences) and analyzed on a LSRFortessa flow cytometer (BD Biosciences). At least 250,000 live events were acquired per sample. Data analysis was performed with FlowJo (TreeStar, Inc.).
Anti cd138 pe
Manufactured by BioLegend
Anti-CD138-PE is a fluorescently labeled antibody that binds to the CD138 (Syndecan-1) surface protein. CD138 is a transmembrane proteoglycan expressed on plasma cells and some epithelial cells. The PE (Phycoerythrin) fluorescent label allows the detection and analysis of CD138-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Fitc anti cd80, by BioLegend (2 mentions)
Cd38 fitc, by BioLegend (1 mentions)
Anti cd16 cd32 fc block, by BD (1 mentions)
Cd19 bv421, by BioLegend (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Ack lysing buffer, by Lonza (1 mentions)
Anti igm apc cy7, by BioLegend (1 mentions)
Anti cd43 pe, by BioLegend (1 mentions)
Anti cd19 fitc, by BioLegend (1 mentions)
Anti mouse cd16 32, by BioLegend (1 mentions)
Anti cd43 pe cy7, by BioLegend (1 mentions)
Anti cd5 apc, by BioLegend (1 mentions)
Anti cd19 v450, by BioLegend (1 mentions)
Anti cd19 apc cy7, by BioLegend (1 mentions)
Anti iga bv421, by BioLegend (1 mentions)
Anti igg2ab bb700, by BioLegend (1 mentions)
Anti igg3 pe cy7, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
7 aminoactinomycin d, by BD (1 mentions)
Streptavidin alexa647, by BioLegend (1 mentions)
6 protocols using anti cd138 pe
1
Bone Marrow Cell Isolation and Analysis
2
Immunophenotyping and Proliferation of B-1 Cells
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For ex vivo analysis of B‐1 cells, 1 × 106 single cells from PL, BM, or spleen were incubated with anti‐mouse CD16/32 (Biolegend) for 15 min on ice for blocking of unspecific Fc‐binding sites. Subsequently, cells were stained with anti‐CD19 FITC, anti‐CD43 PE‐Cy7, anti‐CD5 APC, anti‐IgM APC‐Cy7, and anti‐CD138 PE antibodies (Biolegend and BD Bioscience) for 15 min on ice. To assess cell death, cells were incubated with 7‐aminoactinomycin D (BD Bioscience) before analysis for at least 15 min, but no longer than 60 min.
To analyze proliferation and surface immune globulins after in vitro stimulation, CFSE‐stained cells were incubated with anti‐CD19 V450, anti‐CD43 PE‐Cy7, anti‐CD5 APC, anti‐IgM APC‐Cy7, anti‐IgD‐PerCP, and anti‐IgG1 PE (LPS/IL‐4) or with anti‐CD19 APC‐Cy7, anti‐CD43 PE, anti‐CD5 APC, anti‐IgA BV421, anti IgG2ab BB700, and anti‐IgG3 PE‐Cy7 (LPS only) (Biolegend and BD Bioscience).
All measurements were performed at the BD FACS Canto II and biaxial gating was done with FlowJO Version 10.
To analyze proliferation and surface immune globulins after in vitro stimulation, CFSE‐stained cells were incubated with anti‐CD19 V450, anti‐CD43 PE‐Cy7, anti‐CD5 APC, anti‐IgM APC‐Cy7, anti‐IgD‐PerCP, and anti‐IgG1 PE (LPS/IL‐4) or with anti‐CD19 APC‐Cy7, anti‐CD43 PE, anti‐CD5 APC, anti‐IgA BV421, anti IgG2ab BB700, and anti‐IgG3 PE‐Cy7 (LPS only) (Biolegend and BD Bioscience).
All measurements were performed at the BD FACS Canto II and biaxial gating was done with FlowJO Version 10.
3
Flow Cytometric Analysis of Ca2+ Signaling
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Ca2+ mobilization in YC3.60-expressing cells was analyzed by flow cytometry using CyAn ADP™ (Beckman Coulter) equipped with a 405-nm, solid-state laser as previously described. At 405-nm excitation, FRET intensity was calculated as the ratio of YFP to CFP intensity. Cell sorting was performed using a MoFlo XPD cell sorter (Beckman Coulter) in the Stem Cell Laboratory at our university. Antibodies with the following specificities were conjugated in-house: anti-CD19-Alexa647 and ant-B220-Alexa647. Streptavidin-PE and streptavidin-Alexa647 (which were obtained from BioLegend) were used as secondary reagents. Anti-CD3-PE, anti-CD4-PE, anti-GL-7-APC, anti-B220-Pacific Blue, anti-IgM-PE-Cy7 and anti-CD138-PE were also obtained from BioLegend.
4
Splenocyte Isolation and Phenotyping
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After harvesting the spleen, single-cell suspensions of splenocytes were obtained by filtering through a 70-µm cell strainer. Splenocytes were incubated on ice with CD16/CD32 monoclonal antibody (eBiosience/Thermo Fisher Scientific, Inc.; ready to use; cat. no. 14-0161-85) for 15 min, and then red blood cells were lysed using lysis buffer (BD Biosciences). The splenocytes were then fixed and permeabilized (Fixation/Permeabilization solution; BD Biosciences) before intracellular staining was performed. Cells were stained with the following ready to use antibodies for flow cytometry analysis: anti-CD3 PE-cy7 (eBiosience; Thermo Fisher Scientific, Inc.; cat. no. 25-0032-82), anti-CD4 FITC (eBiosience; Thermo Fisher Scientific, Inc.; cat. no. 11-0041-82), anti-CD8 APC (eBiosience; Thermo Fisher Scientific, Inc.; cat. no. 17-0081-82), anti-CD19 APC-cy7 (eBiosience; Thermo Fisher Scientific, Inc.; cat. no. 47-0193-82), anti-CD138 PE (Biolegend Inc.; cat. no. 142504), and anti-CD69 PE (Biolegend Inc.; cat. no. 104507). FACs data were analyzed using Flowjo software version 10.6 for PC (Tree Star, Inc.).
5
Lupus Spleen Cell Profiling
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Spleens from lupus model were separated in cold RPMI 1640, and then lapped gently and made into signal cell suspension. Cell surface markers was stained with the following fluorochrome-labeled monoclone antibodies (Biolegend, San Diego, CA): PerCP/Cyanine5.5 anti-CD19 (Clone# 6D5)，APC anti-CD69 (Clone#H1.2F3), FITC anti-CD80 (Clone#16-10A1), PE anti-CD86 (Clone#GL-1), and PE anti-CD138 (Clone# 281–2). Then the cells were washed with buffer and identified by flow cytometry (FACS Aria, BD Biosciences, USA), data acquisition were analyzed with FlowJo 7.0 software.
6
Multiparametric Flow Cytometry Analysis
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BD Accuri 6 plus (BD Biosciences) was used for flow cytometry analysis. And we analyzed the results by FlowJo software. Epitope-specific T cells were studied using FITC anti-CD3, APC anti-CD8a and Percp Cy5.5 anti-CD4. Other antibodies used included the following: FITC anti-CD80 (104705, Biolegend), APC anti-CD86 (105011, Biolegend), PE anti-CD62L (104407, Biolegend), APC anti-CD44 (103011, Biolegend), FITC anti-CD103 (121419, Biolegend), PE anti-CD138 (142503, Biolegend), FITC anti-CD45 (103107 Biolegend), PE anti-CD11c (117307, Biolegend). All staining procedures were performed according to the manufacturer's recommendations.
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