17-0699-41

antibody from Invitrogen Antibodies

Targeting: CD69 CLEC2C

Provider product page for 17-0699-41

Flow cytometry

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Antibody data

Antibody Data
Antigen structure
References [22]
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Validations
Flow cytometry [1]
Other assay [20]

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Product number: 17-0699-41 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD69 Monoclonal Antibody (FN50), APC, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The FN50 monoclonal antibody reacts with human CD69, also known as very early activation antigen (VEA). CD69 is approximately 30 kDa and is expressed on the cell-surface as a disulfide-linked dimer. CD69 is rapidly upregulated upon activation and expressed on lymphocytes, monocytes and platelets.
Antibody clone number: FN50
Concentration: 5 µL/Test

CD69 protein structure - 17-0699-41 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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 S-acylation of ZAP-70 is required for T cell activation. A , IL-2 production by P116 (ZAP-70 -/-) Jurkat T cells stably rescued with WT or acylation-deficient C564R ZAP-70. IL-2 concentrations were measured by ELISA in supernatants from resting cells or cells stimulated for 24 h with plate-bound anti-CD3 antibody. Data shown are representative of three independent biological repeats and represented as mean +- SEM. B and C , Expression of CD25 and CD69 T cell surface activation markers by P116 stably rescued with WT or C564R ZAP-70. Cells were stimulated for 24 h with plate-bound anti-CD3 antibody and analyzed by flow cytometry. D and E , Quantification of CD25 and CD69 expression measured by flow cytometry. Data shown are pooled from three independent biological repeats and represented as mean +- SEM.

Supportive validation

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Experimental details: Figure 7 Synergistic combinations avoid global T-cell activation and induction of cytokine release. Resting CD4+ CD25- T-cells were isolated from a HIV-1 negative donor blood. Cells were incubated 24 h with 1 nM bryologs and 100 nM HDAC inhibitor. Cells were stained with fluorescent-conjugated antibodies (a) CD25-APC and (b) CD69-FITC and analyzed by FACS (see Methods). Data indicate mean values and error bars indicate mean +- s.e.m. (n >= 3). ***p < 0.001, (ANOVA). (c) ELISA results for cytokine release from CD4+ CD25- T-cells following 24 h incubation with LRA combinations (see Methods). ( d ) Toxicity profile of LRA combinations. Resting CD4+ T-cells were incubated with compounds at indicated concentrations and MTT toxicity assay was performed as described in Methods.

Supportive validation

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Experimental details: Fig. 1 GTE blocks T cell activation. a CD69 induction decreased in GTE-treated Jurkat T cells. Jurkat T cells were activated by PMA/ionomycin alone (P/I; solid line ) or with 25 mug/mL GTE ( long dashed line ). GTE treatment alone ( short dashed line ) was used as a control. The cells were stained with an anti-CD69-PE antibody. The CD69-positive cells were then analyzed by flow cytometry. Data were assessed with FlowJo software. b Decreased proliferation of GTE-treated PBMCs. Freshly purified PBMCs were pretreated with various doses of GTE for 30 min and then stimulated by 2.5 mug/mL PHA. Cell proliferation was examined by 3 H-thymidine incorporation after a 20 h pulse with 0.5 muCi/well 3 H-thymidine. Data are expressed as counts per minute (CPM) of 3 H-thymidine uptake. A significant difference from the vehicle is indicated as * P < 0.05 or ** P < 0.01

Supportive validation

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Experimental details: Figure 3 hEND-CD3/BiTE activates T cells, stimulates T-cell proliferation, and increases secretion of Th1 cytokines. (A) PBMCs were co-cultured with 293T (grey signal) or 293T-hE (pink signal) cells in the presence of hEND-CD3/BiTE. The expression of T-cell activation markers CD69 (left panels) and CD25 (right panels) was detected in CD4 + (upper rows) and CD8 + (lower rows) by flow cytometry. (B) Proliferation of CFSE-labeled T cells in response to hEND-CD3/BiTE and other indicated reagents in the solid phase was measured using flow cytometry. The proliferation index was calculated using the ModFit LT software. (C) Supernatants were collected from the co-cultures of PBMCs with 293T (white bar) or 293T-hE cells (purple bar) in the presence of hEND-CD3/BiTE. The secretion of Th1 cytokines, including IFN-gamma, and TNF-alpha, and Th2 cytokine IL10 was measured using ELISA assays. * P < 0.001.

Supportive validation

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Experimental details: 10.7554/eLife.44360.007 Figure 3. Lower proliferative responses and CCR2 and CCR5 cell surface levels in activated CD4 +T cells from ECr/VCrs. ( A ) Reduced CD69, but not CD25 levels in activated CD4 +T cells from ECr/VCrs. Graph shows representative data N = 13 (Ctrl), n = 9 (EC/VC) and n = 21 (ECr/VCr). ( B ) Comparable frequencies of naive CD45RA + and memory CD45RO + T cells after anti-CD3/CD28 activation between groups (n = 2 per group). ( C ) CCR5 and CCR2 cell surface levels measured by flow cytometry are reduced in freshly thawed (NS, non-stimulated) and activated CD4 +T cells (anti-CD3/28) from ECr/VCr. ( D ) Percentages of CCR5 +in effector memory (EM) and central memory (CM) compartments of activated CD4 +T cells (n = 2 per group). ( E ) Reduced CCR2 and CCR5 cell surface levels, expressed as MFI, in activated (anti-CD3/28) CD4 +T cells from ECr/VCr. Data in D-E shown pooled results from different experiments with n = 10 (Ctrl and EC/VC) and n = 19 (ECr/VCr). ( F ) Positive correlation between CCR2 and CCR5 cell surface levels. ( G ) Positive correlation observed between ccr2/ccr5 RNA levels and cell surface expression. Values obtained using the non-parametric Spearman correlation test. *p

Supportive validation

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Experimental details: 10.1371/journal.pone.0244771.g004 Fig 4 Celastrol does not activate resting CD4+ T cells. Resting CD4 + T cells were isolated from three healthy donors and treated with 400 nM celastrol, 200 nM JQ1, 400 nM vorinostat, 20 nM romidepsin, or 0.2 ng/ml bryostatin for 24 hours. Expression of CD25 and CD69 was quantified by flow cytometry.

Supportive validation

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Experimental details: Fig. 7 Aberrant cell-to-cell communication signaling associated with therapeutic resistance. a Differentially expressed genes (NR vs. R) in CD4+ and CD8+ T-cells pre- and post-ibrutinib treatment, respectively. Filled circle sizes are proportional to the Log2-scaled fold changes of each gene. Upregulated genes are shown in red; downregulated genes are shown in blue. Pre: pre-treatment; Post: post-treatment. b Representative genes are shown in violin plots. c Alterations (NR vs. R) of ligand-receptor-based cell-to-cell communication networks based in pre- and post-treatment samples. d Flow cytometry validation of upregulated CD69 and CXCR4 expression in ibrutinib nonresponders in comparison to the responders in additional patient cohorts ( n = 65 samples collected from 22 patients). The line in the box is the median value. The bottom and top of the box are the 25th and 75th percentiles of the sample. The bottom and top of the whiskers are the minimum and maximum values of the sample. p Values from the two-side Wilcoxon Signed-rank Test are shown. e Reverse correlation between the proportion of PRF1+ CD8 T cells (cytotoxic) and the expression of CXCR4 using scRNA-seq. The bounds of shape correspond to 95% confidence band for the regression line. The Pearson correlation coefficient ( r ) is shown. p Value corresponds to F test of linear regression model.

Supportive validation

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Experimental details: Figure 2. T cell activation marker CD69 levels, WST-1 proliferation analysis and cell migration of FV treated Jurkat T cells. Cell surface expression of CD69, measured by flow cytometry, following 24 hour treatment with human FV (7 ug/mL) or control (50% (vol/vol) glycerol/H2O) in Jurkat T cells. The T cell mitogen PHA (20 ug/mL) served as positive control for CD69 expression. Left: Relative cell surface expression of CD69. Mean values + SD (n = 6 biological parallels) of two individual experiments. Right: Representative histogram showing the cell surface expression of CD69 for isotype control (background staining), control, human FV, and PHA (positive control). B) Cell viability following 24, 48, and 72 hours of treatment with human FV (7 ug/mL) or control (50% (vol/vol) glycerol/H2O) in Jurkat T cells. WST-1 was added to the cells and OD measurements were conducted after 1 hour incubation. Mean values + SD (n = 15 biological parallels) of three individual experiments. C) The relative number of migrated Jurkat T cells in response to human FV (7 ug/mL) or control (50% (vol/vol) glycerol/H2O) in the bottom chamber of a transwell assay. CXCL12 served as a positive control for migration. Cells were allowed to migrate for 24 hours. Mean values + SD (n = 9 biological parallels) of three individual experiments. P -values for two-group comparisons (Mann-Whitney U) are indicated.

Supportive validation

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Experimental details: FIGURE 1 Distribution of conventional T cells and Tregs in the peripheral blood. (A) Flow cytometry analysis of CD4 + and CD8 + T cells in PBMCs from moderate-to-severe plaque psoriasis patients and healthy controls. (B) Summary plots showing individual results of the frequency of CD4 + and CD8 + T cells in moderate-to-severe plaque psoriasis patients versus healthy controls. (C) Flow cytometry analysis of memory (CD45RO+) and naive (CD45RA+) CD4 + and CD8 + T cells in PBMCs from moderate-to-severe plaque psoriasis patients and healthy controls. (D) Summary plots showing individual results of the frequency of CD45RA+ and CD45RO + CD4 + and CD8 + T cells in moderate-to-severe plaque psoriasis patients versus healthy controls. (E) Flow cytometry analysis of CD69 expression in CD4 + and CD8 + T cells in PBMCs from moderate-to-severe plaque psoriasis patients and healthy controls. (F) Summary plots showing individual results of the frequency of CD69 + CD4 + T cells and CD69 + CD8 + T cells in moderate-to-severe plaque psoriasis patients versus healthy controls. (G) Flow cytometry analysis of CD4 + CD25 + Foxp3+ Tregs in PBMCs from moderate-to-severe plaque psoriasis patients and healthy controls. (H) Summary plots showing individual results of the frequency of CD4 + CD25 + Foxp3+ Tregs in psoriasis patients versus healthy controls. Data show mean +SEM. p -values were determined by paired Student's t-test. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.

Supportive validation

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Experimental details: FIGURE 4 i NKT cells activation status and cytokine production in PBMCs of psoriasis patients. Intracellular IFN-gamma and IL-4 production of circulating i NKT cells was analyzed after stimulation with Cell Stimulation Cocktail for 5 h. (A) Representative FACS dot plots for iNKT cell CD69 expression in psoriasis patients and healthy controls. (B) Summary plots showing individual results of the frequency of CD69 + iNKT cells in psoriasis patients versus healthy controls. (C) Representative histogram and summary plots showing individual results of the MFI of IFN-gamma-producing iNKT cells in psoriasis patients versus healthy controls. (D) Representative histogram and summary plots showing individual results of the MFI of IL-4-producing iNKT cells in psoriasis patients versus healthy controls. (E) Representative histogram and summary plots showing individual results of the MFI of IL-17-producing iNKT cells in psoriasis patients versus healthy controls. (F) Summary plots showing individual results of IL-4-producing i NKT cells/IFN-gamma-producing i NKT cells and IL-17-producing i NKT cells/IFN-gamma-producing i NKT cells in psoriasis patients versus healthy controls. Data show mean +SEM. p -values were determined by paired Student's t-test. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Supportive validation

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Experimental details: Cancer-secreted exosomes reprogram HDLECs to suppress CD8 + T cell immunity (A) Confocal imaging showed the transfer of GFP-labeled exosomes (green) to phalloidin-labeled HDLECs (red) at the indicated time point using Transwell chamber. (B and C) Exosomes secreted by Siha and hCEp were detected by transmission electron microscopy (TEM) (B) and NanoSight analysis (C). Scale bar, 100 nm. (D) Western blot for characteristic proteins of exosomes compared with conditioned media (CM) from Siha and hCEp. (E and F) Flow cytometry analysis of PD-L1 expression (E) and tube-formation assay (F) in HDLECs treated with PBS, hCEp-exo, or Siha-exo. Scale bar, 10 mum. (G-J) Flow cytometry analysis of IFN-gamma (G), CD69 (H), PD-1 (I), and Annexin V (J) expression on CD8 + T cells co-cultured with HDLECs treated with PBS, hCEp-exo, or Siha-exo. (K and L) Quantification of PD-L1 expression (K) and tube formation (L) in HDLECs with indicated treatment. (M-P) Quantification of IFN-gamma (M), CD69 (N), PD-1 (O), and Annexin V (P) expression on CD8 + T cells co-cultured with HDLECs with indicated treatment. The numeric values under the western blot bands represent the protein relative expression (baseline value 1.00). Error bars represent the mean +- SD of three independent experiments. ***p < 0.001.

Supportive validation

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Experimental details: The BNT162b2 mRNA vaccine stimulates the RIG-I/TLR - TNF-alpha - NFkappab axis and activates HIV transcription ex vivo. A qPCR measurements of HIV RNA in supernatants, 48 hours following ex vivo treatment of PBMCs from an ART-treated participant with the indicated concentrations of BNT126b2 (Pfizer BioNTech) or mRNA-1273 (Moderna) mRNA vaccines. B Extension of results from A to n = 6 ART-treated participants, adding treatments with 2% volume/volume Fluzone(tm) influenza vaccine, 25 nM bryostatin-1, 40 nM romidepsin, or 2 mug/ml phytohemagglutinin-L (PHA). P values were calculated by Friedman test with Dunn's multiple comparison test (two-tailed). C Flow cytometry data from the same samples n = 5 donors harvested for ( B ) Shown are % CD69 + (activated) following gating on viable CD4 + T-cells. Data are presented as mean values +/- SD. D Cell-associated HIV RNA measures from the same samples as ( B ) P value of 0.0487 was calculated by a two-tailed paired t test between No Tx and 1% BNT162b2. E - G Bulk mRNA-seq data was generated using a subset of the samples plotted in (WWH-B032, OM5011, and OM5334). B , E Principal component analysis (PCA). The results show that transcriptional profiles of BNT126b2- and mRNA-1273-treated cells are more similar to 'No treatment' and to each other than Fluzone(tm)-treated cells. F Gene set enrichment analyses showing pathways activated following mRNA vaccine treatments. Benjamini-Hochberg corrected P-values were calculated with the fgsea pack

Supportive validation

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Experimental details: Figure 6 RSV-specific CD8+ T cells in BAL display a distinctive resident memory phenotype. Tetramer+ CD8+ T cells in blood and BAL were co-stained for markers to assess their differentiation status. ( a ) CD69 and CD103 as canonical markers of resident memory CD8+ T cells are shown in blood ( n =9) and BAL ( n =5) from infected volunteers. Significant P values for two-tailed Wilcoxon matched pairs tests in blood compared with baseline are shown (day 7, P =0.0313; day 10, P =0.0039; day 14, P =0.0313; and day 28, P =0.0313). ( b ) Memory markers CD45RA and CCR7 are shown in blood ( n =19) and BAL ( n =8). Mean+-significant P values for two-tailed Wilcoxon matched pairs tests compared with baseline are shown in blood for T-effector/effector memory cells (day 7, P =0.0034; day 10, P =0.0002; day 14, P =0.0002; day 28, P =0.0067) and effector memory T cells re-expressing CD45RA (day 7, P =0.0443; day 10, P =0.0025; day 14, P =0.0003; day 28, P =0.0135). ( c ) Proliferation and activation markers Ki-67 and CD38 are shown in blood ( n =19) and BAL ( n =8). Significant P values for two-tailed Wilcoxon matched pairs tests are shown compared with baseline in blood (day 7, P =0.0025; day 10, P =0.0001; and day 14, P =0.0005) and BAL (day 7 versus day 10, P =0.0444; and day 10 versus day 28, P =0.0022 as no Ki-67+CD38+ cells were found in any baseline samples). Throughout, representative plots from a single subject at day 0, 10 and 28 post infection are shown with tetramer+ cells as red

Supportive validation

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Experimental details: Figure 3 Viral MAIT cell activation in vitro . MAIT cells from healthy individuals were analysed by flow cytometry, gated on live CD3 + CD161 ++ Valpha7.2 + cells. ( a , b ) PBMC's ( n =7) were co-cultured with autologous monocyte-derived DC's exposed to DENV (MOI=1) as described in 'Methods'. ( c - e ) CD8 + T cells isolated from PBMC's ( n =11-12) were co-cultured with IAV-exposed macrophages (MOI=1) as described in 'Methods', unless indicated otherwise. ( f ) CD8 + T cells isolated from PBMC's ( n =11-12) were co-cultured with macrophages exposed to the clinical H3N2 influenza A strain (A/England/691/2010 ( n =7)) or influenza B (B/Florida/04/2006 ( n =8)) (MOI=1) as described in 'Methods'. ( g , h ) CD8 + T cells isolated from PBMC's ( n =7-12) co-cultured with macrophages exposed to HCV (MOI=1) as described in 'Methods'. Proportion of MAIT cells producing IFN-gamma ( a , c , d , f , g ), TNF-alpha ( c , d ), CD69 ( c , d ) or Granzyme B ( b , e , h ). ( d ) Representative flow cytometry plots. All data are representative from at least two independent experiments. Bars represent means+-s.e.m. Statistical significance was determined with the Kruskal-Wallis test followed by the Dunns' test ( f ) or the Mann-Whitney test ( a , b , e , g , h ). ns>0.05, * P 0.05, *** P

Supportive validation

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Experimental details: Fig. 4 Active NK cells with a unique transcriptome profile. a Top two enriched gene sets (ranked by normalized enrichment score) of five different datasets from GSEA of the ""Inflamed NK"" cluster compared to the rest of the cells were plotted. b The expression of CD69 in the BM sample was shown as a violin plot. The y -axis represents log-normalized expression value. c Module score was calculated using up-regulated DEGs of ""Active NK"" (left) or ""Inflamed NK"" (right) cluster from BM sample and plotted via violin plots. d Up-regulated IEGs from ""Active NK"" cluster were plotted using heatmap of the BM sample. e The expression of CXCR4 in the BM sample was shown as a violin plot. The y -axis represents log-normalized expression value. f Percentage of CXCR4 + NK cells (gated on Lin - CD56 + cells) was evaluated via flow cytometry. g The expression of CXCR4 in CD57 +/- , CD62L +/- , or NKG2A +/- CD56 dim NK populations from BM was assessed via flow cytometry (top). Percentage of CXCR4 + cells within each population were quantified (bottom). n >= 6 from two to five independent experiments. Paired Student's t test was used for the statistical analysis. * P < 0.05; ** p < 0.01; *** p < 0.001; n.s. stands for ""not significant."" Source data for f and g are provided as a Source Data file. See also Supplementary Fig. 5

Supportive validation

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Experimental details: Figure 1--figure supplement 4. Tat-induced transcriptome changes are also observed at the protein level. Flow cytometry analysis of Jurkat-GFP and -Tat cell lines induced with doxycycline for 24 hr and stained with CD4-APC and CD69-PE antibodies or unstained (negative control) to monitor levels of CD4 and CD69 proteins expressed at the cell surface. Note the increase in the CD69+ population in the presence of Tat. GFP, green fluorescent protein. DOI: http://dx.doi.org/