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9 protocols using anti cxcr5

1

Multiparametric Flow Cytometry of Splenocytes

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Single cell suspensions of splenocytes were resuspended in PBS containing 1% FBS and stained in the dark on ice for 20 minutes. Antibodies used were PeCy7 conjugated anti CD95, PerCP conjugated anti-CD4, PE conjugated anti-PD-1, anti-CD138, and anti-IgD (BD Biosciences); PerCP conjugated anti-CD3 and Pacific Blue conjugated anti-B220 (Biolegend); and Alexa 647 conjugated anti-GL7 (eBioscience). For CXCR5 staining, cells were stained with purified anti-CXCR5 (BD Biosciences) at 4°C for 1 hour in PBS containing 1% FBS, 1% BSA and 2% normal mouse serum (Sigma). Cells were then stained on ice for 30 minutes with biotin conjugated Affini-pure goat anti-rat (Jackson Immunoresearch) followed by staining with APC conjugated streptavidin for 30 minutes on ice.
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2

Comprehensive Immune Profiling of Tumor-Bearing Mice

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The following Abs were used: anti-CTLA-4 (HMCD15201, Thermo Fisher), anti-CD39 (143804, BioLegend), anti-CD8 (553031, BioLegend), anti-CD73 (127220, BioLegend), anti-Tbet (644810, BioLegend), anti-CD44 (103030, BioLegend), anti-KLRG1 (138414, BioLegend), anti-CD11b (101230, BioLegend), anti-CXCR5 (551961, BD Biosciences), anti-CD25 (564571, BD Biosciences), anti-CD4 (553052, BD Biosciences), anti-CD107a/b (553793/558758, BD Biosciences), anti-B220 (561102, BD Biosciences), anti-PD1 (11-9985-81, eBioscience), anti-Foxp3 (50-5773-82, eBioscience), anti-CD11c (17-0114-82, eBioscience), anti-CD45 (12-0451-82, eBioscience), anti-CD49b (25-5971-82, eBioscience) and anti-TCF-1 (2206S, Cell Signaling Technology). Fixable Viability Dye (65-0865-14, eBioscience)-stained cells were excluded from analysis. Tumor-bearing mice were sacrificed; the tumors were sliced and digested with type I collagenase (A004194-0001, Sangon Biotech) for 1 hour at 37°C; and the spleens and dLNs were ground to generate single-cell suspensions. The single-cell suspensions were filtered through 70 µm strainers (352350, BD Biosciences) and stained as described. The stained cells were evaluated by BD FACS Canto II flow cytometry, and the flow cytometry data were analyzed with FlowJo software (Tree Star).
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3

Flow Cytometric Analysis of Lymph Node Cells

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Cells from the two draining LNs of each individual mouse were pooled and stained with fluorescently labeled antibodies to GL7, CD8, B220, TCR-β, CD95 (Fas) (all from BD Biosciences), PD-1, Ter119, GR1, CD11c (all from eBioscience), and CD4 (all from BioLegend). CXCR5 staining was performed using purified anti-CXCR5 (BD Biosciences), followed by FITC anti-rat IgG (Southern Biotech), and normal rat serum (eBioscience). The stained cells were analyzed using a Gallios cytometer (Beckman Coulter) and the generated data analyzed using FlowJo Software (Tree Star).
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4

Comprehensive Murine Immune Cell Analysis

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Anti-CD16/CD32 (BD, 2.4G2 [anti-FcγR]) was used as a blocking reagent and PI-PECF594 (Invitrogen, CAT P3566) was used to identify dead cells. The following antibodies and reagents were used for germinal center B-cell staining: anti-IgD-BV-605 (BioLegend,11-26c.2a), anti-B220-APCeFlour780 (eBioscience, RA3-6B2), anti-CD38-Alexa 700 (eBioscience, 90), and anti-CD95-PE-CY7 (BD, Jo2). For T follicular helper cell (Tfh) staining, anti-CXCR5 (BD, 2G8), anti-rat IgG (fab’) Alexa-647 (Jackson Immuno Research, Code:712-606-153). 2W-specific T-cells were identified using a 2W:I-Ab tetramer labeled with PE (50959 I-A(b)) or BV421(50960 I-A(b) (NIH Tetramer Core Facility), and the following antibodies: anti-CD3-Alexa flour 700 (BioLegend, 17A2), anti-CD4-FITC (BioLegend, RM4-5), anti-B220-APCeFlour780 (BioLegend, RA3-6B2), and anti-PD1-PE-CY7 (eBioscience, J43). The Click-iT™ Plus EdU Alexa fluor 647 Flow cytometry kit was used for EdU staining of newly synthesized DNA (Invitrogen, CAT C10634). Porcine cardiac myosin (Sigma-Aldrich, CAT: M0531) and a cardiac myosin monoclonal antibody (ThermoFisher, CAT: MA1-26180) was used for investigation of myosin-reactive antibody responses.
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5

Multiparametric Flow Cytometry of Murine Splenocytes

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Single-cell suspensions were prepared from spleens by standard gentle mechanical disruption. Antibodies for surface staining, anti-B220 (RA3-6B2), anti-CD44 (IM7), anti-SLAM (TC15-12F12.2), anti-IgD (11-26c.2a) were purchased from Biolegend. Anti-CD95/Fas (Jo2), anti-T-and B-cell activation antigen (GL7), anti-CD19 (ID3), anti-CD4 (RM4-5) were purchased from BD Biosciences. Anti-PD1 (J43) and anti-IgM (Il/41) were purchased from eBioscience. CXCR5 staining was done using either purified anti-CXCR5 or biotin labeled anti-CXCR5 (BD Biosciences) and performed as previously described [51] (link). Anti-CD8a (5H10) and Alexa Fluor 647 labeled streptavidin was purchased from Life Technologies. GC were detected with PNA-FITC (Vector Laboratory) and all surface stains included viability dye, 7-AAD (eBioscience). For intracellular staining, splenocytes were first stained with surface makers followed by staining with live/dead fixable cell staining from Life Technologies. Cells were then fixed and permeabilized using Foxp3 Staining Buffer set purchased from eBioscience following manufacturer's protocol. Cells were stained for intracellular proteins with anti-Bcl-6 (K112-91, BD Biosciences) and anti-Ki67 (B56, BD Biosciences) and Foxp3 (FJK-16s, eBioscience).
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6

Isolation of Tfh, effector, and resting T cells

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One week-old (8 mice/group) and adult CB6F1 mice (6 mice/group) were immunized s.c. at the base of the tail with either HA/CAF01 or HA/GLA-SE. Ten days post vaccination the 2 inguinal draining LNs were collected, samples from each group were pooled and CD4+ T cells were enriched by using the EasySep™ Mouse CD4+ T Cell Isolation Kit (Stemcell Technologies). Total CD4+ T cells were stained with fluorescently labeled antibodies to CD4, B220, PD-1, CXCR5, and streptavidin. CXCR5 staining was performed using purified anti-CXCR5 (BD Pharmingen), followed by FITC anti-rat IgG (Southern Biotech), and normal rat serum (Invitrogen). DAPI was added before sorting as a dead cell discriminator dye.
Highly pure CD4+CXCR5highPD-1high Tfh cells, CD4+CXCR5dimPD-1dim effector T cells and the CD4+CXCR5negPD-1neg resting T cells (non- Tfh) were simultaneously isolated from the enriched CD4+ T cells by flow-cytometry sorting using a MoFlo® Astrios™ flow cytometer (Beckman Coulter); (purity ≥99%).
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7

Multiparameter Flow Cytometry Analysis

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Cell cultures were performed using RPMI 1640 (Mediatech Inc.) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 50 mM 2-mercaptoethanol (Sigma-Aldrich). Fluorescent anti-CD3, anti-CD4, anti-CD8α, anti-CD19, anti-CD25, anti-CD38, anti-CD44, anti-CD62L, anti-CD69, anti-CD80, anti-IgM, anti-IgG, anti-IL-4 and anti-IL-17 antibodies were purchased from Biolegend. Anti-CTLA-4, anti-PD-1, anti-Fas, anti-ICOS, and anti-CXCR5 were purchased from BD Biosciences-Pharmingen. Anti-MHCII, anti-GL7, anti-IFNγ, anti-IL21 and an anti-Foxp3 staining kit were purchased from eBioscience. BD CytoFix/Perm buffer was used for intracellular staining. For cytokine detection, cells were stimulated by Leukocyte Activation Cocktail (BD Biosciences) for 4 to 6 hours and then fixed in eBioscience Fix/Perm buffer for intracellular cytokine staining. Cells were run on a BD LSR II flow cytometer or a Beckman Coulter Navios flow cytometer and analyzed by Flowjo (Flowjo LLC).
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8

Multicolor Confocal Microscopy of Immune Cells

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The following antibodies were used for confocal microscopy at 1:25–1:100
concentrations. Primary antibodies to anti-CD4-alexa fluor 660 conjugated, anti-B220-FITC
conjugated, anti-CD8, biotin labeled-PNA, anti-GL-7, biotin labeled-anti IgD, anti-Ki-67,
anti-Bcl-6, anti-CD80, and anti-Gata-3 were all purchased from eBioscience (San Diego,
CA). Anti-CD11c-FITC and anti-PNAd was purchased from BioLegend (San Diego, CA).
Anti-CXCR5, anti-MHC II (IA/IE), anti-FDC-1, anti-CD138, anti-MAdCAM-1, and anti-CD35 were
all purchased from BD Biosciences (San Diego, CA). Anti-F4/80 was purchased from AbD
Serotec, (Raleigh, NC) anti-phosph-Zap70 was obtained from Cell Signaling Technologies
(Beverly, MA) and anti-Iba-1 was acquired from Wako Chemicals USA (Richmond, VA). Both
anti-CD3 and anti-CXCL13 were obtained from R&D (Minneapolis, MN). The IRBP
p161-180/MHCII/IgG dimer reagent was made in our lab (27 (link)) and is not commercially available.
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9

LCMV-Specific T Cell Characterization

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gp33-H-2Db and np396-H-2Db tetramer production, surface, and intracellular staining, and analysis were performed as previously described [35 (link)]. Intracellular staining for IFN-γ was performed on single cell suspended splenocytes incubated with LCMV specific peptides (gp33 or gp61) for 1 h at 37°C followed by addition of Brefeldin A (eBioscience, San Diego, CA, USA) and subsequent incubation for 5 h at 37°C. After surface staining, cells were fixed with 2% formalin and permeabilized with 0.1% Saponin (Sigma-Aldrich, St. Louis, MO, USA) prior to staining with intracellular antibodies. Gp66-I-A(b) tetramer staining was performed for 30 min at 37°C prior to surface antibody staining at 4°C. Anti-CD3, anti-CD8, anti-IFN-γ, anti-CD4, anti-B220, anti-NK1.1, anti-CD25, anti-CD44, and anti-CD95 were obtained from eBioscience, San Diego, CA, USA. Anti-CXCR5 was obtained from BD Bioscience. PNA was procured from Vector Laboratories, Burlingame, CA, USA.
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