Prior to perfusion, spleens were harvested from experimental mice. Single cell suspensions from spleens were treated with ACK erythrocyte lysis buffer. Mice were perfused with 25 mL of ice-cold PBS and brains and spinal cords were collected from perfused mice and dounce homogenized to obtain single cell suspensions. CNS cells were purified by centrifugation for 30 min in a 30% Percoll (GE Healthcare) solution as previously reported (25 (link)). Cells were incubated with the anti-Fc receptor antibody 2.4G2 prior to the addition of antibodies. The following antibodies were purchased from BD Biosciences: CD45-FITC, CD8α-APC-H7, CD19-APC-H7, CD19-BV510, B220-PE-TxRed, B220–PE-CF594, CD11b-AlexaFluor-700, MHC-v450. The following antibodies were purchased from eBioscience: MHCII-Pacific Blue, CD11c-PECy7. The following antibodies were purchased from BioLegend (San Diego, CA): CD138-PE, MHCII (I-A/I-E)-Pacific Blue, Thy1.1-PerCP, CD4-APC. Cells were acquired on a Gallios flow cytometer (Beckman Coulter) and analyzed with FloJo software (TreeStar) with doublets being excluded.
Cd138 pe
Manufactured by BioLegend
Sourced in United States
CD138-PE is a fluorescently-labeled antibody that binds to the CD138 cell surface antigen. CD138 is a proteoglycan expressed on plasma cells and some epithelial cells. The PE (phycoerythrin) fluorescent label allows for the detection and analysis of CD138-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Cd19 fitc, by BioLegend (2 mentions)
B220 apc, by BioLegend (2 mentions)
B220 bv605, by BioLegend (2 mentions)
Cd19 apc, by BD (2 mentions)
Cd45 fitc, by BD (1 mentions)
Cd11c pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd4 apc, by BioLegend (1 mentions)
Cd19 bv510, by BD (1 mentions)
Cd19 apc h7, by BD (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Cd11b alexa fluor 700, by BD (1 mentions)
Cd8α apc h7, by BD (1 mentions)
Mhc 2 pacific blue, by Thermo Fisher Scientific (1 mentions)
Mhcii 1 a 1 e pacific blue, by BioLegend (1 mentions)
Thy1.1 percp, by BioLegend (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Flojo software, by Tree Star (1 mentions)
Fortessa, by BD (1 mentions)
4 6 diamidin 2 phenylindol dapi, by Thermo Fisher Scientific (1 mentions)
14 protocols using cd138 pe
1
Immune Cell Isolation and Analysis
2
Immunophenotyping of ex vivo B cells
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Freshly isolated or ex vivo cultured human cells were washed in PBS with 5% bovine serum (FACS buffer) and subsequently stained for 30 minutes at 4°C with fluorophore conjugated antibodies against CD19, CD20, CD22, CD27, or IgD (all BD Biosciences). Cells were then washed twice in FACS buffer and analysed on a FACS Canto2 (BD Biosciences). Where appropriate dead cells were excluded using 4,6-Diamidin-2-phenylindol (DAPI; Thermo Fisher).
To analyse B cells after 9–11 days in culture, plates were spun at 400×g for 3 minutes, supernatants were taken for analysis by ELISA and cells in pellets were re-suspended and pooled for each condition. Cells were washed with PBS and stained with NIR fixable live/dead dye (Molecular probes) for 15 minutes at room temperature and washed with FACS buffer (2%BSA, 2mMEDTA, PBS). Live/dead staining was followed by an incubation with Fc Block reagent (BioLegend) for 10 minutes at RT and finally staining with a mix of labelled antibodies: CD45-BV785, CD19-FITC, CD27-BV711, CD38-APC, CD138-PE, and IgD-BV421 (all BioLegend) following the manufacturers recommendations for 30 minutes at 4C. Stained cells were washed and fixed with 2% PFA in PBS for 10 minutes at RT. Stained cells were analysed in a Beckton Dickinson Fortessa with 355, 405, 488, 561, and 633nm lasers.
To analyse B cells after 9–11 days in culture, plates were spun at 400×g for 3 minutes, supernatants were taken for analysis by ELISA and cells in pellets were re-suspended and pooled for each condition. Cells were washed with PBS and stained with NIR fixable live/dead dye (Molecular probes) for 15 minutes at room temperature and washed with FACS buffer (2%BSA, 2mMEDTA, PBS). Live/dead staining was followed by an incubation with Fc Block reagent (BioLegend) for 10 minutes at RT and finally staining with a mix of labelled antibodies: CD45-BV785, CD19-FITC, CD27-BV711, CD38-APC, CD138-PE, and IgD-BV421 (all BioLegend) following the manufacturers recommendations for 30 minutes at 4C. Stained cells were washed and fixed with 2% PFA in PBS for 10 minutes at RT. Stained cells were analysed in a Beckton Dickinson Fortessa with 355, 405, 488, 561, and 633nm lasers.
3
Flow Cytometry Analysis of Immune Cell Subsets
4
Multi-parameter Immune Cell Profiling
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Surface staining was performed in ice-cold PBS with 2% FCS for 15 min in the presence of FcγRII/III blocking antibody (CD16/32). The cells were surface-stained with the relevant antibodies in PBS + 2% FBS for an additional 20 min at 4 °C.
The following fluorochrome-conjugated monoclonal antibodies were used for cell phenotyping: B220 APC (clone RA3-6B2), CD11b PE (clone M1-70), CD138 PE (clone 281-2), CD19 FITC (clone 6D5), CD19 BV421 (clone C068c2), CD206 PE-Cy7 (clone C068c2), CD4 PE (clone RM4-5), CD5 PE-Cy7 (clone 53–7.3), CD8a Ly-2 APC/Fire 750 (clone 53–6.7), F4/80 APC-Cy7 (clone BM8), F4/80 APC (clone BM9), IA-d Alexa Fluor 647 (clone 39–10-8), SIRPα APC (clone 15–414) from Biolegend and CD3 eFlour 660 (clone 17A2). Viability dye eFluor 780 reagent (eBioscience, USA) or propidium iodide were used for live/dead cell discrimination.
Flow cytometry was conducted on a FACSCanto II flow cytometer or FACSAria III Cell Sorter (BD Biosciences), and data analysis was performed using the FlowJo software (Tree Star, Inc., Ashland, OR, USA).
The following fluorochrome-conjugated monoclonal antibodies were used for cell phenotyping: B220 APC (clone RA3-6B2), CD11b PE (clone M1-70), CD138 PE (clone 281-2), CD19 FITC (clone 6D5), CD19 BV421 (clone C068c2), CD206 PE-Cy7 (clone C068c2), CD4 PE (clone RM4-5), CD5 PE-Cy7 (clone 53–7.3), CD8a Ly-2 APC/Fire 750 (clone 53–6.7), F4/80 APC-Cy7 (clone BM8), F4/80 APC (clone BM9), IA-d Alexa Fluor 647 (clone 39–10-8), SIRPα APC (clone 15–414) from Biolegend and CD3 eFlour 660 (clone 17A2). Viability dye eFluor 780 reagent (eBioscience, USA) or propidium iodide were used for live/dead cell discrimination.
Flow cytometry was conducted on a FACSCanto II flow cytometer or FACSAria III Cell Sorter (BD Biosciences), and data analysis was performed using the FlowJo software (Tree Star, Inc., Ashland, OR, USA).
5
Multiparameter Analysis of Immune Cell Subsets
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Single-cell suspensions from spleens, bone marrow, or peripheral lymph nodes were blocked for 30 min using FBS. Cell-surface staining was achieved by incubating cells at 4°C for 30 min with fluorescence-conjugated anti–mouse antibodies (clone; source): XBP1s–Alexa Fluor 647 (Q3-695; BD), XBP1s-phycoerythrin (PE; Q3-695; BD), B220–Alexa Fluor 488 (RA3-6B2; BioLegend), B220-BV605 (RA3-6B2; BioLegend), CD43-PE (eBioR2/60; eBioscience), CD19–Alexa Fluor 647 (6D5; BioLegend), IgM-PE-Cy7 (RMM-1; BioLegend), IgD-FITC (11-26c.2a; BioLegend), GL7-PE (GL7; BioLegend), AA4.1-PE-Cy7 (AA4.1; BioLegend), CD1d-PerCP-Cy5.5 (1B1; BioLegend), CD23-FITC (B3B4; BioLegend), CD3-APC-Cy7 (145-2C11; BioLegend), CD4-BV605 (RM4-5; BioLegend), CD8α-PE-Cy7 (53–6.7; BioLegend), and CD138-PE (281–2; BioLegend). Viability staining was accomplished using DAPI exclusion during acquisition. Acquisition of B, T, and dendritic cell populations was performed on an LSRII cytometer (BD) harboring a custom configuration for the Wistar Institute. Cytometry data were analyzed using FlowJo software (7.6.1; Tree Star Inc.).
6
Identification of B Cell Subsets in Mice
7
Phenotyping B-cell and T-regulatory subsets
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For analysis of B‐cell populations and T regulatory cells, live single‐cell suspensions were prepared from spleens and stained with propidium iodide (PI; catalog number P4864; Sigma/Merck), CD45R/B220‐PE‐Cy7 (1/400; catalog number RA3‐6B2; BioLegend, San Diego, CA, USA), CD138‐PE (1/400; catalog number 281–2; BioLegend), GL7‐Pacific Blue (1/200; BioLegend), peanut agglutinin (PNA)–fluorescein isothiocyanate (1/400; catalog number L7381; Sigma/Merck), IgG1‐APC (1/400; catalog number RMG1‐1; BioLegend), CD4‐Pacific Blue (1/400; catalog number RM4‐5; BD Biosciences), CD25‐APC (1/100; catalog number PC61; BD Biosciences) and FoxP3‐PE (catalog number MF23; BD Biosciences). Antibodies were diluted in FACS buffer (Hanks' Balanced Salt Solution with 2% fetal calf serum). For staining with anti‐FoxP3, cells were fixed and permeabilized with the Mouse FoxP3 buffer set (BD Biosciences). B‐cell populations were defined as follows: plasma cells, B220lowCD138hi; conventional B cells, B220hi CD138low; GC B cells, B220hiGL7hiPNAhi; non‐GC B cells, B220hiGL7lowPNAlow; isotype switched, B220hiGL7hiPNAhiIgG1hi; nonisotype switched, B220hiGL7hiPNAhiIgG1low. T regulatory cells were identified as CD4+CD25+FoxP3+. Cells were analyzed on a BD FACSCanto II (BD Biosciences) after exclusion of PI+ dead cells. Data were analyzed using FlowJo version 10.6.2 cell cycle analysis software (BD Biosciences).
8
Quantifying Splenic Lymphocyte Subsets
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Splenic lymphocytes were collected for flow cytometric analysis. After centrifugation at 300 × g for 10 min at 4 ℃, the supernatant was discarded, and the cells were re-suspended. After adding 10 µL of an Fc receptor blocker (Miltenyi Biotec, 130-09257575), the cells were incubated at 4 °C for 10 min. Subsequently, an allophycocyanin-conjugated antibody, CD19-APC (BD, 550992), and a phycoerythrin-conjugated antibody, CD138-PE (BioLegend, 142504), were added at a volume of 1 µL each, and the cells were incubated at 4 °C for 30 min in the dark. After washing, centrifugation, and resuspension, the samples were analyzed using flow cytometry (Merck Millipore, USA) and IDEAS software (Merck Millipore, USA).
9
Kidney Immune Cell Profiling via Flow Cytometry
10
PBMC Isolation and Immunophenotyping
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Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples with an EDTA tube by density gradient centrifugation using Ficoll–Paque, then resuspended in a freezing solution and stored in liquid nitrogen. Frozen PBMCs were thawed and washed, then stained with fluorescently labeled monoclonal antibodies [anti-human CD3-FITC, CD16/CD56-PE, CD45-PerCP, CD19-APC (Beckton Dickens (BD), MultitestTM), CD19-APC, CD27-FITC, CD24-PerCP, CD38-Alexa Fluor 700, IgD-APC/Cy7, and CD138-PE (Biolegend)] and, then six color immunofluorescence staining was utilized (BD FACS Aria II, Becton Dickinson, Franklin Lakes, NJ). Data were analyzed using Cell Quest (BD) and FlowJo7.6.5 software.
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