Cells to be stained were resuspended in FACS buffer (HBSS containing 1% FCS) and incubated with the indicated antibodies for 15 minutes on ice. Cells were then washed in FACS buffer before acquisition on an LSR-II flow cytometer (BD Bioscience, Franklin Lakes, NJ) and analysis using Flowjo (Treestar). Antibodies (Biolegend, San Diego, CA unless otherwise stated) used were anti-mouse CD4 PerCP-Cy5.5, CD8 Pacific Blue/APC-cy7, PD-1 FITC, CXCR5-biotin (BD Bioscience), CD44 Pacific Blue, GL-7 FITC, FAS PE, CD138 APC, CD19 APC-cy7, CD23 PE, CD21 PerCP-Cy5.5, CD11b-biotin, CD11c Pacific Blue/APC, B220 PE, PDCA-1 Pacific Blue and streptavidin APC/FITC/PerCP. For intracellular staining of markers, an intracellular staining kit (Fix/Perm, eBioscience, San Diego CA) was used together with anti-mouse Foxp3 PE (eBioscience).
Gl7 fitc
Manufactured by BD
The GL7-FITC is a fluorescently labeled antibody used for flow cytometry and immunofluorescence applications. It binds to the GL7 antigen, which is expressed on activated B cells and germinal center B cells. The FITC (Fluorescein Isothiocyanate) fluorescent label allows for the detection and visualization of the GL7-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Fas jo2 pe cy7, by BD (3 mentions)
Ebioscience cell proliferation dye efluor670, by Thermo Fisher Scientific (3 mentions)
Facscanto, by BD (3 mentions)
Cd138 apc, by BD (2 mentions)
Cxcr5 biotin, by BD (2 mentions)
Fas pe, by BD (2 mentions)
B220 pe, by BD (2 mentions)
Cd16 cd32 2.4g2, by BD (2 mentions)
B220 ra3 6b2 apc efluor780, by Thermo Fisher Scientific (2 mentions)
Igd 11 26c fitc, by Thermo Fisher Scientific (2 mentions)
Cd86 po3.1 pe, by Thermo Fisher Scientific (2 mentions)
Hcd2 rpa 2.10 apc, by Thermo Fisher Scientific (2 mentions)
Cxcr4 l276f12 apc, by BioLegend (2 mentions)
Cd86 gl1 percp cy5, by BioLegend (2 mentions)
κ 187.1 fitc, by BD (2 mentions)
Cd19 1d3 apc cy7, by BD (2 mentions)
Igg1 a85 1 fitc, by BD (2 mentions)
Igm 2 41 apc, by Thermo Fisher Scientific (2 mentions)
Cd138 pe, by BD (2 mentions)
Streptavidin percp, by BD (2 mentions)
13 protocols using gl7 fitc
1
Multiparameter Flow Cytometry Analysis
2
Multiparameter Flow Cytometry Analysis
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Single cell suspensions from tissues were ACK lysed and stained in 1% FBS in PBS containing 0.05% sodium azide. Cells were gated according to size and granularity based on FSC-A and SSC-A. Doublets were excluded by FSC-A versus FSC-H gating. Non-antigen-specific binding was blocked with CD16/CD32 (2.4G2) (BD Biosciences). The following antibodies were used for staining: B220 (RA3–6B2) APC-eFluor780, IgM (II/41) APC, IgD (11–26c) FITC, CD86 (PO3.1) PE, hCD2 (RPA-2.10) APC (Thermo Fisher Scientific), CXCR4 (L276F12) APC, CD86 (GL1) PerCP Cy5.5 (BioLegend), κ (187.1) FITC, CD19 (1D3) APC-Cy7, FAS (Jo2) PE-Cy7, GL7 FITC and IgG1 (A85–1) FITC (BD Biosciences). Cell proliferation was analyzed using the eBioscience Cell Proliferation Dye eFluor670 (Thermo Fisher Scientific). Samples were acquired on a FACSCanto (BD Biosciences) and analyzed with Flowjo (Becton, Dickinson and Company).
3
Lymphocyte Isolation and Characterization
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Lymphocytes were isolated as described and stained with anti-mouse IgD PerCP-Cy5.5 (1:400, 405710), LPAM (integrin α4β7) PE (1:100, 120605; (Biolegend, San Diego, CA), B220 V500 (1:400, 561227), CD19 APC-H7 (1:200, 560143), CD80 PE (1:500, 553769), CD273 APC (1:200, 560086), CD138 PE (1:200, 553714), IgM PE-Cy7 (1:200, 552867; BD Biosciences), CCR9 PE-Cy7 (1:100, 25-1991), CD73 PE-Cy7 (1:50, 25-0731), IgA PE (1:50, 12-4204), GL7 eFluor 450 (1:100, 48-5902), CD38 Alexa700 (1:800, 56-0381), CD21/35 Pacific Blue (1:800, 57-0212; eBiosciences) or CCR10 APC (1:100, FAB2815A; R and D systems. Minneapolis, MN) and were analysed using LSR II (BD Biosciences) or Navios (Beckman Coulter, Brea, CA) flow cytometers. For sorting, cells were labelled with anti-mouse CD138 PE (1:200, 553714), CD19 PE-Cy7 (1:200, 552854), CD80 APC (1:200, 560016) and GL7 FITC (1:100, 553666) before sorting using a FACSAria (BD Biosciences). Cells were sorted into tubes that had been coated with 2% BSA/PBS overnight, and pelleted by centrifugation at 600 g before being resuspended in PBS and injected into recipient mice or used for gene sequence analysis.
4
Multicolor Immunofluorescence Staining of Splenic Cells
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Spleens were frozen in OCT (Sakura) and cut into 8-μm-thick sections using a cryostat microtome. The sections were dried overnight, blocked with 5% goat sera (Dako), and stained with the following anti-mouse antibodies: B220-APC (RA3-6B2; BD Biosciences), CD138-PE (281-2; BD Biosciences), GL-7-FITC (BD Biosciences), Ly6G-PE (1A8; BioLegend), hCD2-FITC (RPA-2.10, BioLegend), IgG3-APC (R40-82), and CD11b-FITC (M1/70; BD Biosciences). Images were acquired on a confocal laser scanning microscope (Leica; TCS SP5) and processed with Photoshop software (Adobe Systems). For histopathological evaluation of the joints, paws were removed from the mice at the end point (day 61) and fixed by immersion in 4% paraformaldehyde. Paws were then decalcified in 10% ethylenediaminetetraacetate for 2 to 3 wk at room temperature, followed by paraffin embedding and sectioning. Deparaffinized and rehydrated slides were stained with H&E and used to assess joint inflammation and bone degradation (seven slides per joint at 5- and 200-μm intervals between slides). Images were acquired on a Zeiss Axioplan microscope that was equipped with an Olympus SC30 digital camera.
5
Flow Cytometry for Immune Cell Profiling
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Single cell suspensions of 1×106 cells were washed twice with FACS buffer (2% BSA/2 mM EDTA/PBS, 0.1% NaN3) and maintained in the dark at 4°C throughout experiments. Flow cytometric data were acquired using a CantoII flow cytometer and FACSDiva software (both from BD Biosciences). FlowJo software (Tree Star, Inc.) was used for data analyses.
For marker expression determinations, cells were incubated for 15 min on ice with anti-mouse Abs, including IgG1 (RMG1-1)-FITC, IgD (11-26c.2a)-Pacific Blue (both from Biolegend), CD38 (90)-PE, CD86 (GL1)-eFluor 450, CXCR4 (2B11)-PE, IgM (eB121-15F9)-eFluor 450 (all from eBioscience), B220 (RA3-6B2)-APC/APC-Cy7, CD21 (7G6)-PE, CD23 (B3B4)-PE-Cy7, CD25 (PC61)-APC-Cy7, CD40 (3/23)-FITC, CD95 (Jo2)-PE-Cy7, GL7-FITC, IgD (11-26c.2a)-FITC/PE, IgM (II/41)-FITC/APC/PE-Cy7, IgG3 (R40-82)-biotin, streptavidin-APC (all from BD Biosciences), and PNA-FITC (Vector). Immunostained cells were washed twice in FACS buffer prior to incubation with 7AAD (Sigma). Viable cells were gated from the 7AAD-negative population prior to analysis.
For intracellular staining to detect caspase 3 cleavage, cells were fixed and permeabilized using the BrdU-Flow kit (BD Pharmingen) prior to incubation with PE-conjugated Ab specifically recognizing cleaved caspase 3 (BD Biosciences).
For marker expression determinations, cells were incubated for 15 min on ice with anti-mouse Abs, including IgG1 (RMG1-1)-FITC, IgD (11-26c.2a)-Pacific Blue (both from Biolegend), CD38 (90)-PE, CD86 (GL1)-eFluor 450, CXCR4 (2B11)-PE, IgM (eB121-15F9)-eFluor 450 (all from eBioscience), B220 (RA3-6B2)-APC/APC-Cy7, CD21 (7G6)-PE, CD23 (B3B4)-PE-Cy7, CD25 (PC61)-APC-Cy7, CD40 (3/23)-FITC, CD95 (Jo2)-PE-Cy7, GL7-FITC, IgD (11-26c.2a)-FITC/PE, IgM (II/41)-FITC/APC/PE-Cy7, IgG3 (R40-82)-biotin, streptavidin-APC (all from BD Biosciences), and PNA-FITC (Vector). Immunostained cells were washed twice in FACS buffer prior to incubation with 7AAD (Sigma). Viable cells were gated from the 7AAD-negative population prior to analysis.
For intracellular staining to detect caspase 3 cleavage, cells were fixed and permeabilized using the BrdU-Flow kit (BD Pharmingen) prior to incubation with PE-conjugated Ab specifically recognizing cleaved caspase 3 (BD Biosciences).
6
T-B Cell Activation and Phenotyping
7
Multiparameter Flow Cytometry Analysis
8
Comprehensive Immune Cell Profiling
9
Multicolor Immunofluorescence of Murine Spleen and Kidney
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6μm-8μm sections of optimal cutting temperature (OCT compound, Fisher Healthcare)-embedded murine spleen and kidneys were cut using a cryostat NX (Thermo Scientific) and immediately fixed in acetone before blocking with 5% FBS/PBS. Primary conjugated antibodies (TCRβ-PE, IgD-FITC, CD1d-AF647, CD35-APC, NP-PE, GL7-FITC, CD138-APC, IgG1-FITC; BD Biosciences and BioLegend) were used to label tissue sections, followed by 5% FBS/PBS wash. Coverslips were mounted using Prolong™ Glass Antifade Mount with NucBlue (Invitrogen) and sections were imaged using a Zeiss fluorescent microscope. Analysis included 3-5 images per section, multiple sections per spleen, multiple animals per organ, as noted in figure legends.
10
Visualization and Quantification of Germinal Centers
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