14-9185-82

antibody from Invitrogen Antibodies

Targeting: CXCR5 BLR1, CD185, MDR15

Provider product page for 14-9185-82

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [21]
Comments [0]
Validations
Flow cytometry [1]
Other assay [14]

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Product number: 14-9185-82 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD185 (CXCR5) Monoclonal Antibody (MU5UBEE), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The MU5UBEE monoclonal antibody reacts with Human CD185. CD185, which is also known as C-X-C chemokine receptor 5 (CXCR5) and Burkitt lymphoma receptor 1 (BLR1), is a seven transmembrane G protein-coupled receptor originally identified in Burkitt's lymphoma. In peripheral blood, CXCR5 is expressed on B cells, CD4+ T cells (but not Th1 or Th2 cells), as well as on a subpopulation of memory (CD45RO+) T cells. CXCR5+ circulating T cells are in a resting state and migrate to the lymph nodes due to expression of CCR7 and CD62L. In tonsil, CXCR5 is expressed on nearly all CD4+ cells along with CD45RO and such activation markers as CD69 and ICOS. Tonsillar CXCR5+ cells have been shown to induce antibody production when co-cultured with B cells, thus supporting their role in providing B cell help. Furthermore, this chemokine receptor plays a critical role in lymphocyte trafficking, in particular T cell migration into the B cell follicles of germinal centers in response to CXCL13, making CXCR5 an established marker of follicular helper T cells. Applications Reported: This MU5UBEE antibody has been reported for use in flow cytometric analysis. Applications Tested: This MU5UBEE antibody has been tested by flow cytometric analysis of human peripheral blood cells. This can be used at less than or equal to 0.25 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: MU5UBEE
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: 4° C

CXCR5 protein structure - 14-9185-82 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Example of the staining and gating strategy for PBMC stimulated with Staphylococcal enterotoxin B (SEB). All gates for non-functional markers were defined using fluorescence minus one (FMO) controls whereas gates for functional markers were defined using the unstimulated samples. A : Gating hierarchy to identify NK cells, NKT-like cells, CD4+ and CD8+ T cells, and T FH -like cells. Initial gating is done on FSC-H and FSC-A to discriminate singlets, followed by the exclusion of events collected during a period of time early in collection when fluctuations may occur. In this example, there were no problems of fluctuations and the time gate was minimized to avoid exclusion of any events. Dead cells and monocytes are excluded by an amine reactive dye and the CD14 marker in the same dump channel. Lymphocytes are gated using FSC-A and SSC-A. Subsequent gating discriminates two subsets of NK cells by CD56 and CD3 expression (CD56dimCD3- and CD56hiCD3-) and NKT-like cells (CD56+CD3+). Within the gate of lymphocytes, CD3+ cells are identified, followed by identification of CD4+ and CD8+ T cells. Of note, NKT-like cells are not excluded from classical T cells and therefore are overlapping populations. Finally, T FH -like cells are identified as CXCR5+ CD45RA- CD4+ T cells that have a low expression of CCR7 and are PD-1+. B : Functional markers for CD4+ and CD8+ T cells. A gate is applied for each cytokine, not taking into account the coexpression of other markers. Boolean gates are the

Supportive validation

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Experimental details: Figure 4 Extended Immunogen Release Induces Higher nAb Titers Than Conventional Immunization (A-E) Immunogen doses of 100 or 20 mug s.c. immunizations of BG505 SOSIP.664. (A) BG505 nAb titers at week 26 (n = 6 or 12). (B) BG505 SOSIP binding titers at week 26 (n = 6 or 12). (C) Kinetics of BG505 nAb titers. (D and E) GC B cell (D) and GC Tfh cell (E) frequencies after the first, second, and third immunizations. (F-L) Bolus (conventional) versus continuous immunogen delivery of BG505 SOSIP.v5.2 immunogen. (F) Immunization schedule and sampling for continuous antigen delivery using osmotic pumps. (G) BG505 nAb titers in animals immunized by osmotic pump (red) or conventional bolus (Conv, black) ( * p < 0.05; ** p < 0.01; n = 6). (H) Peak BG505 nAb titers after the third immunization (n = 6). (I and J) GC B cell (I) and GC Tfh cell (J) frequencies after the first, second, and third immunizations. (K) Proliferation of GC Tfh cells at week 11. Flow cytometry was gated on CXCR5 hi PD-1 hi GC Tfh cells. (L) Frequency of Ki67 + GC Tfh cells at week 11 (n = 12). All nAb titer and ELISA binding Ab data represent geometric mean titers with geometric SD. All cell-frequency data represent the mean and SD. See also Figure S4 .

Supportive validation

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Experimental details: Figure 4 Unchanged cTfh cells and altered Ig profile in sporotrichosis patients. (A) Statistical graphs in the upper row were for CD4 + CXCR5 + Tfh while the lower row were for CD4 + CXCR5 + PD1 + Tfh. The comparison of cTfh percentages were between HC (n = 24) and whole patients (n = 50), patients with different duration (< 6 mon, n = 24; > 6 mon, n = 26) and clinical types (FF, n = 33; LF, n = 17). (B) Comparison of serum levels of total IgG, IgM, and IgE between patients (n = 46) and HC (n = 24). (C, D) Distribution of IgG subtypes (IgG1, IgG2, and IgG3 and IgG4) in patients (in whole: n = 20; SD: n = 7; LD: n = 13) and HC (n = 19). Error bars represent mean+-SD. * P < 0.05, ** P < 0.01, and NS P >= 0.05.

Supportive validation

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Experimental details: Figure 1 Temporal and spatial dynamics of CXCR5+ NK cells in peripheral lymph nodes during primary SIVagm infection (A) Gating strategy of CXCR5+ NK cells in pLN. NK cells were gated as CD45 + CD3 - CD20 - NKG2A/C + CXCR5 + cells as previously reported (; , p. 80). A representative image is shown. The example corresponds to pLN cells from an African Green Monkey infected by SIVagm at day 9 post-infection. (B) Kinetic of CXCR5 + NK cell percentages in pLN during SIVagm infection, dpi = days post-infection. Each green circle indicates an individual animal (depending on the time point, n = 3 or 6 AGM); p value = *

Supportive validation

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Experimental details: Figure 3 CXCR5+ NK cell induction and gene expression profile (A) Staining of NK cells co-cultured with autologous B cells separated by a transwell membrane illustrating CXCR5 expression on NK cells. The CD3 labeling is used here as a negative control. (B) Dot plot representative of CXCR5+ NK cells from uninfected AGM after 6 days of culture: NK cells cultured alone and NK cells co-cultured with autologous B cells separated by a transwell membrane. (C) The fold change of CXCR5+ NK cell frequencies among total NK cells compared with the frequency of CXCR5+ NK cells in NK cells cultured alone is shown after 6 days of co-culture. B and NK cells were isolated from PBMC of 6 non-infected AGMs (p value = *

Supportive validation

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Experimental details: Figure 2. Ggg inhibits the migration of P2RY8-expressing cells. (a) P2RY8 ligand bioassay using the indicated concentrations of Ggg, glutathione (GSH), GG-PP, or LTC4 with 50 ng/mL CXCL12 (n=4 biological replicates). (b, c) Transwell migration assays of tonsil cells towards CXCL12 mixed with the indicated concentrations of Ggg. Left plots, representative flow cytometry of CD19 + cells showing the gate for CD38 + IgD - GC B cells (b) or of CD4 + cells showing the gate for PD-1 + , CXCR5 + Tfh cells (c). Right graphs show summary data for indicated cell types. (n=3 tonsils, 2 technical replicates each). (d) Internalization assay using cells expressing OX56 epitope-tagged P2RY8, read by measuring surface OX56 levels (representative flow cytometry histogram, left). Right graphs show summarized data for the indicated receptors (n=6 biological replicates, one-way ANOVA with Bonferroni's multiple comparisons test). Data are pooled from 3 experiments (a,b,c,d). Graphs depict mean with s.d.

Supportive validation

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Experimental details: Figure 2 9-Day Transduction and Expansion Protocol Yields Sufficient Cells for Infusion, Which Are Viable and Maintain Co-expression of Two Transduced Genes (A) Total number of cells in culture on days 5 and 9 of the expansion protocol and (B) viability of the transduced cells using PBMCs from seven different animals. Cell number and viability was monitored by trypan blue exclusion counting on the countess cell counter. (C) Representative flow plots showing gating strategy and expression of both the CD4-MBL CAR and CXCR5 in transduced cells on day 9. (D) Expression of CAR, CXCR5, and co-expression of CAR and CXCR5 on days 5 and 9 in cells from seven different animals. The bars represent the mean value and 95% confidence interval.

Supportive validation

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Experimental details: Figure 3 Treatment of Macaques with Antiretroviral Drugs Prior to Collection of PBMCs Leads to a Reduction of Transduction Efficiency with Gammaretroviral Vectors PBMCs from six animals using cells collected prior to treatment with anti-retroviral drugs (Pre-ART) or during antiretroviral treatment (ART) were used in the 9-day transduction and expansion protocol. On day 9, cells were evaluated for expression of MBL, representing the CAR, and CXCR5 by flow cytometry. A Wilcoxon matched pairs signed rank test was used to determine significance. Colors denote cells from six individual animals. The bar represents the median value.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. Longitudinal Sampling of GCs by LN FNA after eOD-GT8 60-mer Immunization (A) Schematic of LN FNA and blood sampling after IM (left) or s.c. (right) immunization. (B) Flow cytometry identification of B GC cell frequencies in the ipsilateral (draining) and contralateral (non-draining) LNs after s.c. and IM immunization. B GC cells are KI67 + BCL6 + . Full gating is Figure S2 . (C) Weekly sampling of B GC cell frequency after one immunization. *p < 0.05, **p < 0.01 (paired t test, one tailed). (D) Flow cytometry identification of GC-T FH cell frequencies in the ipsilateral (draining) and contralateral (non-draining) LNs after s.c. and IM immunization. GC-T FH cells are CXCR5 + PD1 + . (E) Weekly sampling of GC-T FH cell frequency after one immunization. Gated on CD4 + T cells. Each point represents an individual LN FNA sample. n = 8, four LN FNAs per immunization condition at each time point. See also Figures S2 and S3 .