PBMC were isolated from peripheral blood by Ficoll gradient and frozen for batched analysis. We designed six multicolor flow cytometry panels to quantify 60 T cell subsets along with two B cell subsets, and calculated the CD4+/CD8+ T cell ratio (Supplementary Table 1 ). The following fluorochrome-conjugated anti-human antibodies were used from BD Biosciences (San Jose, CA): CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CCR4-PE, CD27-PE, CD28-BV421, CD138-BV421, CCR6-BV421, CXCR3-PE, CCR7-A700, IL-17-BV786, IFN-γ-PE-Cy7, iso IgG1k-FITC, iso IgG1k-PE-Cy7, iso IgG2bk-APC, iso IgG1k-APC-Cy7, and iso IgG1k-BV510; from Biolegend (San Diego, CA): CD127-FITC, CD27-APC, CD57-PerCp-Cy5.5, CD19-BV510, PD-1-APC-Cy7, CXCR5-FITC, and TNFα-FITC; from eBioscience (San Diego, CA): CD4-PE-Cy7, and IL-2-PE; from Miltenyi Biotec (San Diego, CA): CD25-APC and KLRG1-PE; from Beckman Coulter (Brea, CA): CD38-PE-Cy7.
Cd138 bv421
Manufactured by BioLegend
Sourced in United States
CD138-BV421 is a fluorochrome-conjugated monoclonal antibody that specifically binds to the CD138 cell surface antigen. CD138 is a transmembrane heparan sulfate proteoglycan that is expressed on plasma cells and some other cell types. This product can be used for the detection and analysis of CD138-expressing cells by flow cytometry or other immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BD (2 mentions)
Cxcr3 pe, by BioLegend (2 mentions)
Ifn γ pe cy7, by BioLegend (2 mentions)
Cd27 pe, by BioLegend (2 mentions)
Cd4 apc cy7, by BioLegend (2 mentions)
Cd28 bv421, by BioLegend (2 mentions)
Cd8 bv510, by BioLegend (2 mentions)
Cd45ro fitc, by BioLegend (2 mentions)
Cd45ra apc, by BioLegend (2 mentions)
Cd45ra apc cy7, by BioLegend (2 mentions)
Ccr4 pe, by BioLegend (2 mentions)
Ccr6 bv421, by BioLegend (2 mentions)
Il 17 bv786, by BioLegend (2 mentions)
Cd38 pe cy7, by Beckman Coulter (2 mentions)
Zinc formalin fixative, by Merck Group (2 mentions)
B220 af488, by Thermo Fisher Scientific (2 mentions)
Cd4 af594, by BioLegend (2 mentions)
Gl7 af647, by BD (2 mentions)
Cd3 percp cy5, by BD (1 mentions)
Cd4 apc, by BioLegend (1 mentions)
7 protocols using cd138 bv421
1
Multicolor Flow Cytometry for T and B Cell Subsets
2
Immunophenotyping of Whole Blood Samples
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Venous blood was drawn into an ethylenediaminetetraacetic acid (EDTA) S-monovette (Sarstedt, Nümbrecht, Germany) for immunophenotyping. Whole blood (100 µl per flow cytometry panel) was directly stained in FACS tubes with fluorescence-labeled antibodies (BD-Biosciences, Franklin Lakes, United States: CD8-PerCP-Cy5.5, CD5-PE, CD45RA-PE-CF594, CD19-PE-Cy5, CD4-PE-Cy7, CD45RO-APC, CD38-A700, CD3-APC-H7, CD138-BV421, CD10-BV510, CD27-BV605, CD14-PE-CF594, CD19-PE-Cy5, CD25-PE-Cy7, CD16-A700, CD3-APC-H7, CD11b-BV421, HLADR-BV510; Biolegend, San Diego, United States: CD11c-BV605; Miltenyi Biotec, Bergisch-Gladbach, Germany: BDCA1-APC, BDCA2-PE) for 15 min in the dark at room temperature (RT). After washing, red blood cell (RBC) lysis was performed with 2 ml ACK lysing buffer (Thermo Scientific, Waltham, United States) for 7 min in the dark at RT. 2 ml of FACS buffer (phosphate buffered saline, 3% fetal calf serum, 1% sodium azide) was added to wash the samples twice. Subsequently, cells were resuspended in 400 µl FACS-buffer. After the staining procedure, cells were measured by flow cytometry (LSR Fortessa cytometer, BD Biosciences, Franklin Lakes, United States) and analyzed with FACS-Diva Software (BD Biosciences, Franklin Lakes, United States). The gating strategy is available in the supplementary data section of this paper (Supplementary Figure S7 ).
3
Spleen Tissue Immunostaining and Imaging Protocol
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After harvest, spleens were passed through a sucrose gradient (10% for 1
h, 20% for 2 h, 30% for 3 h) and flash frozen in Tissue Freezing Medium (General
Data TFM-5) using a Gentle Jane Snap Freezer. 10 μm sections were cut and
dried for 45 min in a 37 °C dry incubator. The sections were soaked in
ice-cold Zinc Formalin Fixative (Sigma) for 15 min at −20°C then
washed in 1× PBS (3 min/ wash). Sections were blocked with 1% BSA in
1× PBS for 2 h at 25 °C. After a 5-min wash in 1× PBS,
sections were stained with the primary antibodies (in 1% BSA in 1× PBS)
for 24 h at 4 °C. Sections were washed in 1× PBS (3 min/ wash)
then mounted using hard set mounting medium for fluorescence (VECTASHIELD
H-1400). Sections were stained with B220-AF488 (5 μg/ml; clone
RA3–6B2, eBioscience), CD4-AF594 (10 μg/ml; clone GK1.5,
BioLegend). GL7-AF647 (6.67 μg/ml; BD Pharmingen), and CD138-BV421 (1.67
μg/ml; clone 281–2, BioLegend). Imaging was done using a Zeiss
LSM710 confocal microscope and processed using IMARIS x64 software (version
9.2.1).
h, 20% for 2 h, 30% for 3 h) and flash frozen in Tissue Freezing Medium (General
Data TFM-5) using a Gentle Jane Snap Freezer. 10 μm sections were cut and
dried for 45 min in a 37 °C dry incubator. The sections were soaked in
ice-cold Zinc Formalin Fixative (Sigma) for 15 min at −20°C then
washed in 1× PBS (3 min/ wash). Sections were blocked with 1% BSA in
1× PBS for 2 h at 25 °C. After a 5-min wash in 1× PBS,
sections were stained with the primary antibodies (in 1% BSA in 1× PBS)
for 24 h at 4 °C. Sections were washed in 1× PBS (3 min/ wash)
then mounted using hard set mounting medium for fluorescence (VECTASHIELD
H-1400). Sections were stained with B220-AF488 (5 μg/ml; clone
RA3–6B2, eBioscience), CD4-AF594 (10 μg/ml; clone GK1.5,
BioLegend). GL7-AF647 (6.67 μg/ml; BD Pharmingen), and CD138-BV421 (1.67
μg/ml; clone 281–2, BioLegend). Imaging was done using a Zeiss
LSM710 confocal microscope and processed using IMARIS x64 software (version
9.2.1).
4
Multicolor Flow Cytometry of PBMC
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We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4+, CD8+, and CD19+. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 106 events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo® software.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4+, CD8+, and CD19+. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 106 events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo® software.
5
Plasmacytoid Dendritic Cell Characterization
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MM-pDCs were co-cultured either in DCP-MM medium (Mattek Corp. Ashland, MA) or complete RPMI-1640 medium supplemented with IL-3 (Peprotech Inc., Rocky Hill, NJ, USA). CD3-PE/FITC/APC; CD4-FITC/PE or APC-Cy7; CD8-APC/FITC, CD56-PE; CD123-PE/PE-Cy5/FITC; and CD138-FITC/PE/APC were obtained from BD Biosciences (San Jose, CA). BDCA-2-FITC and CD11c-APC were obtained from Miltenyi Biotec (Auburn, CA); CD303-, CD304-, CD107a, CD138-BV421, and PD-L1-BV421 were purchased from Biolegend. Immunomagnetic separation kits were purchased from Miltenyi Biotec. The CellTrace Violet and CellTracker Violet/Green flow assay kits were obtained from Life Technologies (USA). CD73 blocking antibody (anti-human CD73 Abs) and TLR-7 agonist were obtained from eBiosciences and Invivogen, respectively. WST-1 Cell Proliferation Reagent was purchased from Clontech Laboratories, Inc. (USA).
6
Spleen Tissue Immunostaining and Imaging Protocol
7
Infection and Differentiation of Primary B Cells
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Purified CD19+ B cells from healthy donors’ PBMCs were infected with DENV-1 at MOI of 5 for 90 min, washed and cultured in the presence of CD40L (0.25 μg/ml; ITS Vietnam), IL-2 (1 ng/ml; Peprotech, NJ, USA), and IL-21 (50 ng/ml; Peprotech) for 6 days. The cells were harvested and stained with Zombie Aqua (AmCyan) viability dye (BioLegend) for live/dead cell gating followed by antibodies CD19 PE/Cy7 (clone HIB19), CD20 PerCp/Cy5.5 (clone 2H7), CD27 APC/Cy7 (clone O323), CD38 APC (clone HB7) and CD138 BV421 (clone MI15), and the percentages of CD27+CD38+ plasmablasts and CD27+CD138+ plasma cells were determined in uninfected and infected B cells with or without stimulation.
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