25-0399-41

antibody from Invitrogen Antibodies

Targeting: ENTPD1 ATPDase, CD39, NTPDase-1, SPG64

Provider product page for 25-0399-41

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [23]
Comments [0]
Validations
Flow cytometry [1]
Other assay [13]

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Product number: 25-0399-41 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD39 Monoclonal Antibody (eBioA1 (A1)), PE-Cyanine7, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The eBioA1 monoclonal antibody reacts with human CD39 also known as ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) or NTPDase. CD39 is an integral membrane protein with two transmembrane domains and exists as a homotetramer. It is the most prominent ectoenzyme of the immune system. The function of CD39 is to effectively remove toxic extracellular ATP by converting it to ADP or AMP. CD39 is thought to work together with CD73 to hydrolyze ATP and has been well characterized on Langerhans cells. Expression of CD39 was originally identified on activated lymphocytes. Expression is also found on a subset of T cells, B cells and dendritic cells as well as weak staining on monocytes and granulocytes. Recently, CD39 and CD73 have been found on regulatory T cells (Treg). Expression of CD39 on Treg may facilitate their entry into inflamed areas where high levels of ATP are present. Expression of CD39 on Foxp3+CD4+ cells ranges from 25-45%. Applications Reported: This eBioA1 (A1) antibody has been reported for use in flow cytometric analysis. Applications Tested: This eBioA1 (A1) antibody has been pre-titrated and tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at 5 µL (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. Light sensitivity: This tandem dye is sensitive photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL cell sample + 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 488-561 nm; Emission: 775 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: eBioA1 (A1)
Vial size: 25 Tests
Concentration: 5 µL/Test
Storage: 4° C, store in dark, DO NOT FREEZE!

ENTPD1 protein structure - 25-0399-41 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
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: Figure 5 Induction of CD39+ T reg differentiation by plasma-derived exosomes. ( A ) Expression of CD39 after incubation with total exosome fraction of patients of all stages ( B ) Expression of CD39 after incubation with total exosomes of low stage vs. high stage patients. ( C ) Expression of CD39 after incubation with CD45(-) and CD45(+) exosomes of low stage and high stage patients. Note that only incubation with CD45(-) exosomes showed significant stage-dependent differences. ; n = 18. p values were determined with Mann-Whitney test, with * p < 0.05, ** p < 0.005, **** p < 0.0001. Bars represent standard error of mean (SEM). ( D ) Representative flow cytometry plots depicting CD39 expression of CD4+ T cells after incubation with CD45(-) (left) or CD45(+) (right) exosomes of UICC low stage (top) or high stage (bottom) patients.

Supportive validation

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Experimental details: Figure 5 B cells were harvested after 2 days of co-culture with either NC or HNSCC exosomes or PBS and stained for FACS analysis. ( A ) Frequency of CD39 + CD73 + regulatory B cells. ( B ) The expression of CD39 on B cells was reduced after co-culture with NC or HNSCC exosomes. ( C ) Expression of CD73 on B cells. ( D ) Expression of CD86 on B cells. ( E ) The expression of CD19 on B cells was increased by stimulation with CD40L and IL-4. **: p < 0.01; *: p < 0.05, n = 8 (HNSCC), n = 6 (NC), n = 5 (Unstim). Unstim = Unstimulated B cells, NC = no cancer (exosomes from blood plasma of healthy volunteers), HNSCC, exosomes from blood plasma of HNSCC patients.

Supportive validation

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Experimental details: Figure 7 Effects of TEX on protein expression and functions of T cells. In ( a ) down-regulation of CD69 protein expression on the surface of responder CD4 + Tconv after co-incubation with TEX. Activated CD4 + Tconv were co-incubated with TEX (10 ug protein) produced by the PCI-13 cells or with PBS for 12 h. The CD69 expression levels on CD4 + Tconv were then determined by flow cytometry (MFI) and were converted into MESF units based on calibration curves established with fluorescent calibration beads. The bar graphs show data (mean values +- SD) from 3 independent experiments performed with CD4 + Tconv obtained from different normal donors. The asterisks indicate p values at p < 0.0005. In ( b ) changes in expression levels of CD39 protein on the surface of resting CD4 + CD39 + Treg co-incubated with TEX produced by the PCI-13 cell line or DEX. The exosomes were used at the concentration of 10 ng protein/ assay. Exogenous ATP was added as described in Methods. Flow cytometry ( right ) shows up-regulation of MFI for CD39 in a representative experiment, and the bar graph summarizes results of three experiments performed with Treg obtained from different donors. In ( c ), Production levels of 5' AMP, adenosine and inosine by resting CD4 + CD39 + Treg co-incubated with TEX produced by the PCI-13 cell line. The data are from one of two experiments performed in the presence of exogenous ATP. The analyte levels were measured by mass spectrometry as described in Methods.

Supportive validation

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Experimental details: Fig. 4 Analysis of tumor-infiltrating lymphocytes. a Proportions of CD8 + and CD4 + T cells from biopsy material, and proportions of these that were positive for PD-1 and CD39. Sample UM22 was derived from a patient that has previously been treated with chemotherapy, possibly affecting TIL proportions in this sample. b Assessment of T-cell reactivity against MART-1, gp100 and NY-ESO-1 in yTIL cultures. Proportions found to be reactive are indicated. Samples tested were those with the HLA-A*02:01 genotype, as this genotype is known to present MART-1 and gp100. Samples with this genotype (Supplementary Data 7 ) that are not shown were also tested and found to be negative. c Analysis of relative levels of PDCD1 (PD-1) and ENTPD1 (CD39) expression among different CD8 + T-cell clonotypes, determined by single-cell RNA-seq of yTILs. Clonotypes with one pair of alpha and beta chain were included, and the ones with greatest expression of both markers are highlighted. Point sizes are proportional to clonotype frequency. Gray color indicates clones that were negative for either PDCD1 or ENTPD1 , whereas other colors indicate different clonotypes that correspond to those in Supplementary Fig. 7e . d Expression of T-cell markers and checkpoint receptors in bulk RNA-seq data from biopsies (batch-corrected log 2 RPKM normalized values). yTILs young TILs, TILs isolated and expanded from a biopsy with a low dose of IL-2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Changes in TIL composition after OX40 administration. TIL from a pretreatment biopsy and a surgical specimen after OX40 therapy were assessed for lymphocyte composition and activation markers. The gating strategy is outlined in Supplementary Fig. 3 . a Percentages of CD4+ Tconv cells, CD4+ Treg cells, and CD8+ T cells in each patient before and after OX40 administration, N = 17 patients. b tSNE analysis of the pre and post specimens from patient HNOX07, gated on CD3+ cells. Blue represents the baseline sample, orange the day of surgery sample and gray is the concatenated file. The red circle indicates the population of cells expressing both CD103 and CD39. tSNE analysis was performed on N = 4 patients, one representative patient is shown here, two more patients are shown in Supplementary Fig. 4 . c Flow cytometric analysis of the expression of CD103 and CD39 in CD4+ Tconv cells, CD8+ cells, and CD4+ Treg cells in one immune-responding head and neck squamous cell carcinoma (HNSCC) patient pre- and post OX40 therapy. d Summary of the flow cytometric analysis in (c), left panel depicts CD8+ CD103+ CD39+ T cells and the right panel depicts CD4+ CD39+ T cells; patients with an increase are shown on the left, patients with a decrease are on the right. e Expression of Ki-67 was assessed among memory CD4+ TIL and CD8+ TIL subsets (DN, SP, and DP) in biopsy (pre) and DOS (post) tissue ( N = 17 patients). Blue histograms indicate pre, red indicate post tissues. The left graph sh

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. T cell receptor (TCR) clusters in CD39 + PD1 + tumor-infiltrating lymphocytes (TILs). ( a ) The experimental workflow. ( b-g ) TCRbeta repertoire analysis for CD8 + ( b, d, f ) and CD4 + ( c, e, g ) double-positive (DP) and non-DP TIL subsets sorted from metastatic lymph nodes of eight melanoma patients. Panels show repertoire clonality calculated as [1 - Normalized Shannon-Wiener index] ( b, c ), normalized counts ( d, e ), and cumulative frequency of cluster-related clonotypes, that is, total weight of all clusters as a proportion of TCRbeta repertoire ( f, g ). Paired t-test. ( h ) TCRbeta clusters identified in repertoires obtained from fresh-frozen tumor (FFT) samples, and sorted CD8 + DP and non-DP TILs for HLA-A*02 patient mp26. VDJdb-matched TAA-specific clusters are colored in red. ( i , j ) Cumulative frequency of ( i ) VDJdb-matched TAA-specific clonotypes and ( j ) VDJdb-matched TAA-specific cluster-related clonotypes within CD8 + DP, CD8 + non-DP, and FFT TCRbeta repertoires of patient mp26. One-way ANOVA, Bonferroni multiple comparisons correction. ( k , l ) Proportion of CD137 + cells among CD8 + T cells ( k ) and proportion of VDJdb-matched TAA-specific clonotypes in sorted CD137 + CD8 + T cells ( l ) in DP and non-DP TILs from patient mp26 that were cultured and re-stimulated with TAA-loaded or control dendritic cells. Figure 2--figure supplement 1. ( a ) Fluorescence-activated cell sorting (FACS) gating for sorting of CD8 + and CD4 + CD39 + PD-1 +

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 2 Effect of early ARV initiation on immune activation. (A and E) Gating strategy used in flow cytometry to define HLA-DR + CD8 and CD4 T cells in both whole blood and MLNs. (B and F) Percentages of activated HLA-DR + CD8 and CD4 T cells among total CD8 and CD4 T cells in whole blood and MLNs. (C and G) Gating strategy used in flow cytometry to define CD39 + CD8 and CD4 T cells in both whole blood and MLNs. (D and H) Percentages of activated CD39 + CD8 and CD4 T cells among total CD8 and CD4 T cells in whole blood and MLNs.