Surface staining was done by incubating fresh PBMC with a cocktail of antibodies containing 5 μl CD19 PerCP (Clone 4G7, BD, Catalog No. 345778), 3 μl CD21 PE-CY7 (Clone B-ly4, BD, Catalog No. 561374), 20 μl CD27 FITC (Clone Ll28, BD, Catalog No. 340424), 3 μl CD23 BV421 (Clone M-L233, BD, Catalog No. 562707), 10 μl sIgD PE(Clone IA6–2, BD, Catalog No. 555779), 10 μl sIgM APC (Clone G20–127, BD, Catalog No. 551062) in panel one and 5 μl CD19 PerCP (Clone 4G7, BD, Catalog No. 345778), 3 μl CD21 PE-CY7 (Clone B-ly4, BD, Catalog No. 561374), 20 μl CD27 FITC (Clone Ll28, BD, Catalog No. 340424), 10 μl CD 5 PE(Clone Ll7F12, BD, Catalog No. 345782), 3 μl CD38 APC (Clone HB7, BD, Catalog No. 345807) and 3 μl CD24 BV421 (Clone ML5, BD, Catalog No. 562789) in panel two. Incubations were done for 20 min at room temperature in the dark. Then cells were washed and re-suspended in FACS (Fluorescence Activated Cell Sorting) buffer. Data were acquired on a BD FACSCanto II with Diva software and analyzed using FlowJo software (Version 9.6.2).
Cd27 fitc
Manufactured by BD
Sourced in United States
CD27-FITC is a fluorescently labeled antibody that binds to the CD27 antigen expressed on the surface of certain types of immune cells. It is used in flow cytometry applications to identify and enumerate cell populations expressing the CD27 marker.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 fitc, by BD (6 mentions)
Cd3 percp, by BD (5 mentions)
Facscanto 2, by BD (4 mentions)
Cd19 pe cy7, by BD (4 mentions)
Cd8 apc h7, by BD (4 mentions)
Ccr7 pe, by BD (4 mentions)
Cd3 bv421, by BD (3 mentions)
Cd19 apc, by BD (3 mentions)
Cd38 pe, by BD (3 mentions)
Cd4 apc h7, by BD (3 mentions)
Cd197 pe, by BD (3 mentions)
Il 17 apc, by R&D Systems (3 mentions)
Cd25 pe cy7, by BD (3 mentions)
Cd56 apc, by BD (3 mentions)
Cd80 pe, by BD (3 mentions)
Cd24 pe, by BD (3 mentions)
Cd45ro apc, by BD (3 mentions)
Cd38 pe cy5, by BD (3 mentions)
Cd45ra apc, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
27 protocols using cd27 fitc
1
Comprehensive B-cell Immunophenotyping
2
Isolation and Activation of Naive B Cells
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Freshly obtained peripheral mononuclear cells (PBMCs) were isolated after performing Ficoll density gradient centrifugation. Naive B cells were isolated from PBMCs using the MACS naive B cell isolation kit II in an Automacs (Miltenyi Biotech). Naive B cell enrichment was assessed using CD19 PerCP-Cy5.5, CD27 FITC, CD3 APC (BD Biosciences) and IgD PE (Southern Biotech). We resuspended 1 × 10 5 purified naive B cells in 200 µl RPMI-1640 supplemented medium (10% fetal calf serum, 100 U/ml penicillium, 100 µg/ml streptomycin and 2 mM glutamine). Cells were activated for 4 days with 2 μg/ml of MegaCD40L soluble human recombinant (Enzo Life Sciences, Farmingdale, NY, USA) and 100 ng/ml of human recombinant IL-21 (Gibco, Life Technologies, Carlsbad, CA, USA); unstimulated samples were performed in parallel. Cell cultures were maintained for 4 days at 37°C in a 5% CO 2 atmosphere. Cell activation was measured with the monoclonal antibody combinations CD19 PerCP-Cy5.5, CD27 FITC, CD69 PE (clone L78) and CD86 APC (clone 2331 Fun-1) (BD Biosciences). Responsiveness to stimulation was calculated as the fold increase of MFI in CD69 and CD86 in stimulated cells to the same markers in the unstimulated cells (fold change = MFI-stimulated/MFIunstimulated). The analysis was performed by flow cytometry using FACS Canto II (BD Biosciences) and the FlowJo Software (LLC).
3
Immune Reconstitution Monitoring by Flow Cytometry
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Hematopoietic donor cell chimerism was performed as previously described [15 (link)]. Immune reconstitution was monitored in peripheral blood mononuclear cells (PBMCs) by flow cytometry in between week 0–38. PBMCs were isolated by density gradient centrifugation using Biocoll cell separation solution (Biochrom, Berlin, Germany). Immune cells were stained using the directly fluorescence labeled mAbs CD138-PE (clone: MI15, BD Biosciences, Franklin Lakes, NJ, USA), CD138-PE (clone: MI15, BD Biosciences), CD38-PerCP (clone: HB-7, BD Biosciences), CD19-PacificBlue (clone: SJ25C1, BD Biosciences), CD27-FITC (clone: M-T271, BD Biosciences), IgA-APC (clone: 97924, R&D Sytems, Minneapolis, MN, USA), IgG-APC (clone: 97924, R&D Systems), IgM-APC (clone: IL/41, ThermoFisher), HLA-A2-APC (clone: BB7.2, ThermoFisher), HLA Class I B7-PE (clone: BB7.1, abcam, Cambridge, UK), HLA-Class1 BW6-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) and HLA-Class1 B8-APC (Miltenyi Biotec). Dead cells were excluded from analysis by LIVE/DEAD™ Fixable Aqua Dead Cell Stain (ThermoFisher, Waltham, MA, USA). Measurements were performed using a FACS Canto II or FACS Fortessa (BD Biosciences) and data analyzed using the software FlowJo V10 (FlowJo LCC, Ashland, OR, USA).
4
Quantification of Lymphocyte Subsets in PBMC
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PBMC were isolated by density gradient centrifugation on Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) as described previously [5] (link). Frequencies of lymphocyte subsets were estimated in PBMC by means of flow cytometry using mAb against CD19-PE.Cy7, CD4-APC, CD27-FITC, CD10-APC, IgD-PE, IgM-PE.Cy5, IgG-APC and CD138-APC (BD Pharmingen, San Jose, CA, USA). Non-specific fluorescence was established with APC-mouse IgG1κ, PE-Cy7-mouse IgG1κ, FITC-mouse IgG1κ, PE-mouse IgG2aκ, PE-mouse IgG1κ isotype controls. At least 200,000 events per sample were collected in a FACSAria II flow cytometer (BD Immunocytometry Systems) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Lymphocytes were identified by forward and side-angle light scatter characteristics. At least 80,000 lymphocyte-gated cells were analyzed for each sample. Absolute numbers were estimated based on the frequencies of the cells in flow cytometry and lymphocytes in the differential leukocyte count (Sysmex K-800 Automated Hematology Analyzer, Sysmex America, Inc., Mundelein, IL, USA), and expressed as the number of cells per microliter of blood.
5
Immunophenotyping of PBMCs in RRMS
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For cross-sectional study, fresh peripheral blood mononuclear cells (PBMCs) were surface stained with monoclonal antibodies against CD19-APC-cy7, CD27-FITC, CD24-BV421, CD38-BV510, and PD-L1(CD274)-PE-cy7 (BD Biosciences). For longitudinal study, frozen PBMCs, collected from 11 RRMS patients undergoing alemtuzumab at baseline and 6, 9, and 12 months, were surface stained with monoclonal antibodies as stated above.
6
Plasmablast B cell isolation protocol
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Fresh PBMC were used to sort human plasmablast B cells as described before [12 (link)]. Cells were stained with the following antibody panel: CD3 BV421 (BD Biosciences), CD19 APC (BD Biosciences), CD27 FITC (BD Biosciences), CD38 PE Cy7 (BD Biosciences), CD20 PE (BD Biosciences). Plasmablasts were defined as CD3−/CD19+/CD27+/ CD38+/CD20− and sorted on a FACS Jazz sorter in single cell mode into a 96 well plate.
7
Multiparametric Flow Cytometry Profiling
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Purified peripheral blood mononuclear cells were stained for 30 min at 4°C with the following panel of mouse anti‐human antibodies: CD19 PE‐Cy7 (1:25 dilution), IgG V450 (1:20), CD27 FITC (1:25), CD38 PE (1:25), IgA biotin (1:100, all from BD Biosciences) and CD21 APC (1:20; eBioscience). 7AAD (1:200; Life Technologies) was used to exclude dead cells. Biotin‐labeled antibodies were detected with streptavidin‐Qdot605 (1:100; Life Technologies). BD Bioscience LSRII and Aria I instruments were used for flow cytometric analyses, and Aria II was used for single‐cell sorting.
Human lung A549 epithelial cells, human nAECB primary epithelial cells, and human THP‐1 monocytes were harvested from culture dishes using either trypsin–EDTA or 3% EDTA and stained with the following anti‐human antibodies: CD89 PE (1:10; Biolegend), CD32 PE (1:10; Biolegend), CD16 PE (1:10; BD Biosciences), and CD64 PE (1:10; BD Biosciences). PE‐labeled mouse anti‐human IgG1 κ and IgG2 κ antibodies (Biolegend) served as isotype controls.
Human lung A549 epithelial cells, human nAECB primary epithelial cells, and human THP‐1 monocytes were harvested from culture dishes using either trypsin–EDTA or 3% EDTA and stained with the following anti‐human antibodies: CD89 PE (1:10; Biolegend), CD32 PE (1:10; Biolegend), CD16 PE (1:10; BD Biosciences), and CD64 PE (1:10; BD Biosciences). PE‐labeled mouse anti‐human IgG1 κ and IgG2 κ antibodies (Biolegend) served as isotype controls.
8
MHC Tetramer-Based T-Cell Immunophenotyping
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MHC class I tetramer was prepared in-house. Surface staining of T cells was achieved by addition of tetramer to whole blood, incubation for 10 minutes at room temperature, followed by addition of antibody co-stains for 20 minutes. Whole blood was preferred to thawed PBMCs for tetramer labelling due to greater consistency and signal intensity. Following lysis of erythrocytes using BD FACS Lysing solution (BD Biosciences), cells were either fixed using 2% formaldehyde (v/v) or permeabilized using the BD Cytofix/Cytoperm kit for intra-cellular staining. The following antibodies were used for surface and intra-cellular staining: CD3-PerCP, CD8-Horizon V500, CD8-APCH7, Ki-67-FITC, Bcl-2-PE, HLA-DR-Horizon V450, HLA-DR-PerCP, Perforin-FITC, Granzyme B-Horizon V450, CCR7-PE, CD27-Horizon V450, CD27-FITC, CD28-PECy7, CD28-PE (all BD Biosciences) and CD38-PECy7 (eBioscience), Granzyme B-PE (Caltag), Granzyme K-PE (Santa Cruz), CD45RA-FITC (Beckman Coulter), PD-1-PE and 2B4-PerCPCy5.5 (Biolegend). Intra-cellular cytokine staining with IFN-γ-FITC, TNF-α-APC, and IL-2-PerCpCy5.5 (all BD Biosciences) was undertaken after in vitro stimulation of PBMCs using peptide for 6 hours. Flow cytometry analysis was performed BD LSRII and BD FACSCanto flow cytometers. Flow cytometry data were analyzed using FlowJo software.
9
Multiparameter flow cytometry for cTfh subsets
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PBMCs were stained with CD4-FITC, CXCR5-PE, CXCR3-APC, CCR6-PE, CD27-FITC, CD38-PE, CD40L-PE, and IFNγ-PE antibodies (BDPharmingenTM, USA) and incubated at 4°C for 30 min in the dark. After the antigen–antibody incubation was completed, the specimen was cleaned 3 times with 2.5 mL of flowing washing solution (Hyclone, USA)/PBS, then resuspended in 500 μL of PBS, and put in storage at 4°C for performing upper flow cytometry analysis. The isotype control antibody was used to adjust the compensation of each channel and set the gate parameters. FlowJo software (Version 7.6.1, Tree Star Inc., USA) was employed to analyze the data obtained by flow cytometry. CD4+CXCR5+CXCR3+CCR6 indicates Th1-like cTfh cells, CD4+CXCR5+CXCR3-CCR6 indicates Th2-like cTfh cells, and CD4+CXCR5+CXCR3-CCR6+ indicates Th17-like cTfh cells.
10
Flow Cytometric Analysis of B Cell Subsets
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