cd69 monoclonal antibody (fn50)  (Thermo Fisher)

Journal: iScience

Article Title: Enhanced and sustained T cell activation in response to fluid shear stress

doi: 10.1016/j.isci.2024.109999

Figure Lengend Snippet: Stimulation with FSS in cone-and-plate viscometers induces MSC-mediated calcium influx and does not compromise T cell viability (A) Schematic of cone-and-plate viscometer used to apply FSS treatments. (B) Annexin V/Propidium Iodide (AV/PI) flow cytometry assay of T cells exposed to 5 dyn/cm 2 FSS for 1 h in cone-and-plate viscometers (500,000 cells/mL) with no treatment, with αCD3/CD28 beads, and with GsMTX-4. Data are presented as the percentage of viable cells (AV − /PI − ) alongside representative flow cytometry plots. ( N = 3; paired t test, two-tailed, α = 0.05). (C and D) Intracellular calcium measured immediately (0 days) post activation via flow cytometry in the (C) absence and (D) presence of GsMTX-4. Data are presented as Fluo-4 MFI relative to static control T cells ( N = 3, repeated measures (RM)-one-way ANOVA, α = 0.05) and representative flow cytometry plots of Fluo-4 fluorescence (presented with raw MFI). (E) MFI of forward scatter (FSC-H) and side scatter (SSC-H) measured via flow cytometry in T cells 0–10 days post-activation (day 0, 2, 5, 7: N = 4, day 1: N = 3; day 3, 10: N = 5). (F) CD4/CD8 ratio of T cells 3 days and 7 days post-activation treatments ( N = 4; two-tailed ratio paired t test, α = 0.05, violin plots represent the mean as a solid line and first and third quartiles as dashed lines). Data represent the mean ± SEM unless otherwise stated, of T cells from N independent experiments, where each point within a condition represents a unique donor. All data and statistical analysis related to this figure are provided in Table S1 .

Article Snippet: For flow cytometry: CD69 Monoclonal Antibody (Host: Mouse; clone: FN50) - FITC , BD Biosciences , Cat#:555530; RRID: AB_395915.

Techniques: Flow Cytometry, Two Tailed Test, Activation Assay, Fluorescence

Journal: iScience

Article Title: Enhanced and sustained T cell activation in response to fluid shear stress

doi: 10.1016/j.isci.2024.109999

Figure Lengend Snippet: FSS in the presence and absence of CD3/CD28 antibody-coated beads enhances and sustains the phosphorylation of key activation proteins (A and B) Phosphorylation of NF-κB MFI (relative to static control T cells) at 0–10 days post-activation via flow cytometry. (C and D) Phosphorylation of NF-κB MFI (relative to static control T cells) at (C) 0 days and (D) 3 days post-activation with sheared conditions treated with GsMTX-4 where indicated. FSS conditions without GsMTX-4 are the same points shown in A and B and are included for clarity. (E) Representative confocal images of the phosphorylation of NF-κB 3 days post-activation (phos-NF-κB = green; F-actin = magenta; DAPI = blue). (F and G) Quantitative analysis of the phosphorylation of NF-κB (F) 0 days and (G) 3 days post-activation via confocal microscopy (0 days: N = 2, n = 75; 3 days: N = 3, n = 115) (H and I). Representative (H) confocal images and (I) quantitative analysis of the phosphorylation of c-Fos immediately (0 days) post-activation ( N = 3, n = 175; phos-c-Fos = yellow; F-actin = magenta; DAPI = cyan). (J–L) Representative (J) confocal images and quantitative analysis of (K) nuclear NFATc1 immunofluorescence and (L) NFATc1-DAPI colocalization (%) immediately (0 days) post-activation ( N = 3, n = 120; NFATc1 = yellow; F-actin = red; DAPI = blue). (A–D) Data represent the mean ± SEM of T cells where each point within a condition represents a unique donor. Mean MFI are presented within each bar. Statistics calculated by (A and B) RM one- or (C and D) two-way ANOVA ( α = 0.05) and Tukey’s multiple comparisons post-hoc test. (F, G, I, K, L) Boxplots expressing the median (central line), mean (plus symbol), first and third quartiles (bounds of box), and the highest and lowest values (whiskers), where N = independent experiments and n = total cells analyzed per condition. Statistics calculated by ordinary one-way ANOVA ( α = 0.05). (E, H, J) Scale bar, 10 μm ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. All data and statistical analysis related to this figure are provided in Table S2 .

Article Snippet: For flow cytometry: CD69 Monoclonal Antibody (Host: Mouse; clone: FN50) - FITC , BD Biosciences , Cat#:555530; RRID: AB_395915.

Techniques: Activation Assay, Flow Cytometry, Confocal Microscopy, Immunofluorescence, Expressing

Journal: iScience

Article Title: Enhanced and sustained T cell activation in response to fluid shear stress

doi: 10.1016/j.isci.2024.109999

Figure Lengend Snippet: FSS in the presence of anti-CD3/CD28 antibody-coated beads enhances and sustains the expression of T cell activation markers CD69 and CD25 (A and B) Percentage of T cells expressing CD69 at 0–10 days post-activation via flow cytometry. (C and D) Percentage of T cells expressing CD25 at 0–10 days post-activation via flow cytometry. (E and F) Percentage of T cells co-expressing CD69 and CD25 at 0–10 days post-activation via flow cytometry. (G) Representative flow cytometry plots of CD69 and CD25 expression at 3, 5, 7, and 10 days post-activation. (H and I) Representative (H) flow cytometry plots and (I) quantification of CD69 and CD25 co-expression at 5 days post-activation with sheared conditions treated with GsMTX-4 were indicated. (A–F) Data represent the mean ± SEM of T cells where each point within a condition represents a unique donor. Statistics calculated by RM-two-way ANOVA ( α = 0.05) and Tukey’s multiple comparisons post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. All data and statistical analysis related to this figure are provided in Table S3 .

Article Snippet: For flow cytometry: CD69 Monoclonal Antibody (Host: Mouse; clone: FN50) - FITC , BD Biosciences , Cat#:555530; RRID: AB_395915.

Techniques: Expressing, Activation Assay, Flow Cytometry

Journal: iScience

Article Title: Enhanced and sustained T cell activation in response to fluid shear stress

doi: 10.1016/j.isci.2024.109999

Figure Lengend Snippet: FSS stimulation increases lytic granule production in Pan-CD4 + /CD8 + and CD8 + -isolated primary T cells (A and B) CD107a + percent expression 0–10 days post-activation via flow cytometry. Data represent the mean ± SEM of CD107a + in Pan-T cells isolated from unique donors (day 0, 1, 5: N = 6, day 3, 7, 10: N = 5, day 2: N = 3; RM-two-way ANOVA, α = 0.05). Representative flow cytometry plots of CD107a + expression at 0, 3, 5, and 10 days post-activation relative to static control T cells (presented with raw MFI). (C) Flow cytometry gating strategy for isolated CD8 + T cells. (D–F) CD8 + T cells evaluated in flow cytometry for (D) CD107a + , (E) granzyme B + , and (F) perforin + at 3 days post-activation. Data represent the mean ± SEM and representative flow cytometry plots (presented with raw MFI) of CD8 + T cells isolated through magnetic separation from 3 unique donors ( N = 3; RM-two-way ANOVA, α = 0.05). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. All source data and statistical analysis related to this figure are provided in Table S4 .

Article Snippet: For flow cytometry: CD69 Monoclonal Antibody (Host: Mouse; clone: FN50) - FITC , BD Biosciences , Cat#:555530; RRID: AB_395915.

Techniques: Isolation, Expressing, Activation Assay, Flow Cytometry

Journal: iScience

Article Title: Enhanced and sustained T cell activation in response to fluid shear stress

doi: 10.1016/j.isci.2024.109999

Figure Lengend Snippet: FSS stimulation increases pro-inflammatory cytokine production (A) IFN-γ, TNF-α, and IL-2 quantified via ELISA assay 3 days post-activation. Cell-conditioned media was collected from cultured T cells and the quantified cytokine secretion was normalized to cell number at the time of supernatant collection. Data represent the mean ± SEM of IFN-γ, TNF-α, and IL-2 of T cells isolated from 3 different donors ( N = 3; RM-two-way ANOVA, α = 0.05). (B and C) IFN-γ, TNF-α, and IL-2 MFI (relative to static control T cells) measured in flow cytometry at (B) 3 and (C) 7 days post-activation. Data represent the mean ± SEM of IFN-γ, TNF-α, and IL-2 MFI of T cells isolated from 5 donors ( N = 5; two-tailed ratio paired t test, α = 0.05). Representative flow cytometry plots are presented with raw MFI. Source data and statistical analyses are provided in Table S5 .

Article Snippet: For flow cytometry: CD69 Monoclonal Antibody (Host: Mouse; clone: FN50) - FITC , BD Biosciences , Cat#:555530; RRID: AB_395915.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Cell Culture, Isolation, Flow Cytometry, Two Tailed Test

Journal: iScience

Article Title: Enhanced and sustained T cell activation in response to fluid shear stress

doi: 10.1016/j.isci.2024.109999

Figure Lengend Snippet: Mechanical stimulation via FSS enhances T cell proliferation (A and B) Quantification of (A) proliferating cells (%) and (B) representative flow cytometry plots of violet proliferation dye (VPD450) at 3 days post-activation. Data represent the mean ± SEM of proliferating T cells isolated from 3 to 4 healthy donors, where the mean percentage is presented within the bars (Static, Shear, Static+αCD3/CD28, Shear+αCD3/CD28: N = 4; Shear+GsMTX-4, Shear+αCD3/CD28+GsMTX-4: N = 3; two-way ANOVA, α = 0.05). (C and D) Ki67 + percent expression at 0–10 days post-activation via flow cytometry. Data represent the mean ± SEM of Ki67 + expression in T cells isolated from 4 to 6 donors (day 0: N = 6, day 1: N = 6, day 2: N = 5, day 3: N = 6, day 5: N = 5, day 7: N = 6, day 10: N = 6; RM-two-way ANOVA, α = 0.05). (E) Representative confocal images of T cells 3 days post-activation (Ki67 = yellow; Actin = red; DAPI = blue). Scale bar, 10 μm. (F and G) Quantitative analysis of confocal images representing (F) nuclear Ki67 immunofluorescence and (G) Ki67-DAPI colocalization (%) in T cells 3 days post-activation ( N = 3, n = 150 per group; one-way ANOVA, α = 0.05; Outliers removed, ROUT Method, Q = 1%). Boxplots expressing the median (central line), mean (plus symbol), first and third quartiles (bounds of box), and the highest and lowest values (whiskers), where N = independent experiments and n = total cells analyzed per condition. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. All data and statistical analysis related to this figure are provided in Table S6 .

Article Snippet: For flow cytometry: CD69 Monoclonal Antibody (Host: Mouse; clone: FN50) - FITC , BD Biosciences , Cat#:555530; RRID: AB_395915.

Techniques: Flow Cytometry, Activation Assay, Isolation, Shear, Expressing, Immunofluorescence

Journal: iScience

Article Title: Enhanced and sustained T cell activation in response to fluid shear stress

doi: 10.1016/j.isci.2024.109999

Figure Lengend Snippet:

Article Snippet: For flow cytometry: CD69 Monoclonal Antibody (Host: Mouse; clone: FN50) - FITC , BD Biosciences , Cat#:555530; RRID: AB_395915.

Techniques: Confocal Microscopy, Recombinant, Flow Cytometry, Staining, Electron Microscopy, Cell Isolation, Enzyme-linked Immunosorbent Assay, Software, Microscopy

Journal: Nature Nanotechnology

Article Title: Programmable multispecific DNA-origami-based T-cell engagers

doi: 10.1038/s41565-023-01471-7

Figure Lengend Snippet: a , Treatment schedule of binding and activation trials. Experimental procedure to measure the in vivo binding and activation behaviour on tumour (NALM-6) and T cells. Colour scheme: midnight blue, PTE-2×19-3; teal, PTE-3; mocha, PTE-2×19; red, chassis; violet, Blina-BS. Doses were between 10 and 15 pmol. NALM-6-GFP-luc, PBMC and samples were intravenously administered at the indicated time points. Three mice were injected per group. After 4 h, the mice were sacrificed and sample distribution in the bone marrow was analysed using flow cytometry. b , c , Mean fluorescence intensity (MFI) measurements (left) and representative histograms (right) of flow cytometric measurements of Cy5 fluorescence ( b ) or CD19-BV785 fluorescence ( c ) on NALM-6-GFP-luc cells (indicating Cy5-labelled PTE bound to the tumour cells). d , e , Measurement of CD69 fluorescence on either CD4 + ( d ) or CD8 + ( e ) T cells. In b – e , data are mean ± s.e.m. from n = 3 mice. f , Schematic of the long-term treatment with PTEs in vivo. g , Luminescence images of mice injected with luciferin to visualize NALM-6-GFP-luc tumour cells. The white cross indicates the mouse that was censored due to non-tumour-related toxicities. h , Quantification of bioluminescence measurements. For g and h , n = 5 mice per group; for h , mean ± s.e.m. is shown. For all the panels, statistical significance was calculated using ordinary one-way or two-way analysis of variance with Tukey’s multiple comparisons correction.

Article Snippet: CD69 monoclonal antibody (FN50) , Thermo Fisher.

Techniques: Binding Assay, Activation Assay, In Vivo, Injection, Flow Cytometry, Fluorescence

Journal: Nature Nanotechnology

Article Title: Programmable multispecific DNA-origami-based T-cell engagers

doi: 10.1038/s41565-023-01471-7

Figure Lengend Snippet:

Article Snippet: CD69 monoclonal antibody (FN50) , Thermo Fisher.

Techniques:

Journal: Journal of Cancer

Article Title: In Vitro Effects of Low-energy Ultrasound Treatment on Healthy CD3/CD8+ Lymphocytes, Red blood cells, Acute Myeloid leukemia cells, and Jurkat cell line

doi: 10.7150/jca.83050

Figure Lengend Snippet: CD69-CD137 and CD8+ Granzyme B expression analysis : 106 lymphocytes UT and 2T were incubated after US treatment with 40 U/ml IL-2 and with CD69, CD3 antibodies (A) and with 40 U/ml IL-2 and 15 U/ml IL-7, CD137, CD8 antibodies (B) in presence or absence of CD3/CD28 Dynabeads, and then analyzed by Flow cytometry. Correspondent isotype controls were used to set correct gating. Live lymphocytes (106 cells) were then identified by FSC-H/SSC-H parameters; singlets were gated by FSC-A/FSC-H. (C) For Granzyme B detection 106 lymphocytes, control and 2T, were incubated with 40 U/ml IL-2 and 15 U/ml IL-7 in presence or absence of CD3/CD28 Dynabeads, then lymphocytes were stained for CD8 surface marker followed by Fix and Perm protocol for Granzyme B staining. The density plots are representative of three healthy donor samples. 20000 events were acquired.

Article Snippet: Lymphocytes were also stained with CD8+ activation marker CD69 Monoclonal Antibody (FN50), PE, eBioscience, CD137 (4-1BB) Monoclonal Antibody (4B4 (4B4-1)), PE, eBioscienceTM and its isotype control Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), PE, eBioscience.

Techniques: Expressing, Incubation, Flow Cytometry, Control, Staining, Marker

Journal: International Journal of Oncology

Article Title: Isolation and analysis of tumor-derived extracellular vesicles from head and neck squamous cell carcinoma plasma by galectin-based glycan recognition particles

doi: 10.3892/ijo.2022.5423

Figure Lengend Snippet: EVs functional assay of T cells and PBMCs activation. (A) A total of 30 out of 200 µ l eluted patient plasma EVs from EXÖBead ® isolation and PEG EVs were treated with CD4 + T cells in anti-CD2/3/28 antibodies activation condition. The Violin plot shows that CTLA4 + CD69 Neg T cells emerged only when treated with eluted patient plasma EVs from EXÖBead ® , PEG EVs and T cells activation. Significance was calculated by non-parametric Kruskal-Wallis test with Dunn's multiple comparison test. (B) a total of 5×10 7 particles of eluted patient or control plasma EVs from EXÖBead ® isolation were treated with 1×10 6 PBMCs (ratio: 50:1) under anti-CD2/3/28 antibody activation conditions. Violin plot showed that CD69 + PD-L1 + live CD4 + T cells were derived from treatment with elution buffer alone, from plasma EVs from HNSCC patients (n=13, with technological triplicate) and from EVs from healthy controls (n=3, with technological triplicate). Significance was calculated by Brown-Forsythe and Welch's ANOVA test with Dunnett's T3 multiple comparisons test. EV, extracellular vesicle; PBMC, peripheral blood mononuclear cells.

Article Snippet: After two washing steps, cells were stained with 100 µ l of 1:100 antibody master mix, CD4 Monoclonal Antibody eFluor ® 450 (Clone: SK3, Thermo Fisher Scientific, Inc.), APC anti-PD-L1 antibody: MIH1 (Thermo Fisher Scientific, Inc.), PE anti-human CD279 (PD-1) Antibody: EH12.2H7 (Biolegend Inc.) and CD69 Monoclonal Antibody FITC: FN50 (Thermo Fisher Scientific, Inc.) for 30 min at 4°C.

Techniques: Functional Assay, Activation Assay, Isolation, Derivative Assay

Journal: eLife

Article Title: Phenotypic analysis of the unstimulated in vivo HIV CD4 T cell reservoir

doi: 10.7554/elife.60933

Figure Lengend Snippet: Figure 4. Comparison of kNN latent cells reactivatable by different stimulations and from blood versus tissues using longitudinal specimens. (A) Atlas (gray) and kNN latent cells (red) from three blood specimens from the same donor (PID5003) spaced 2–3 months apart, where reactivation was induced by either PMA/ionomycin or Romidepsin/PEP005. Arrows highlight regions that harbor kNN latent cells in the PMA/ionomycin but not Romidepsin/ PEP005 samples. Red numbers indicate the number of kNN latent cells in each plot. (B) kNN latent cells reactivatable by PMA/ionomycin at two different time points are more similar to each other than to those reactivatable by Romidepsin/PEP005 at the same time point. The empirical cumulative distribution of median distances between kNN cells was calculated between (1) Time point 1 PMA/ionomycin and Time point 2 PMA/ ionomycin samples (red), and (2) Time point 2 Romidepsin/PEP005 and Time point 3 Romidepsin/PEP005 samples (pink), and (3) Time point 2 PMA/ ionomycin and Time point 2 Romidepsin/PEP005 samples (blue). (C) Atlases of memory CD4+ T cells from fine needle aspirates (FNAs) or the blood of the same donor (PID3010) with corresponding kNN latent cells (red). Red arrows point to regions of the FNA tSNEs that are concentrated in kNN latent cells. Bottom plots show each FNA atlas overlaid onto the blood atlas. Red numbers indicate the number of kNN latent cells in each plot. (D) Dot plots demonstrating that FNA but not blood kNN latent cells (red dots) express uniformly high levels of CD27, CD69, CXCR5, PD1, and ICOS. Atlas cells are Figure 4 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.60933 16 of 34 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody OX40 (mouse monoclonal) Fluidigm Cat#3158012B (1 mg/100 mL) Antibody CCR7 (mouse monoclonal) Fluidigm Cat#3159003A (1 mg/100 mL) Antibody CD28 (mouse monoclonal) Fluidigm Cat#3160003B (1 mg/100 mL) Antibody CD45RO (mouse monoclonal) Biolegend Cat#304239 (1 mg/100 mL) Antibody CD69 (mouse monoclonal) Fluidigm Cat#3162001B (1 mg/100 mL) Antibody CRTH2 (rat monoclonal) Fluidigm Cat#3163003B (1 mg/100 mL) Antibody PD-1 (mouse monoclonal) Biolegend Cat#329941 (1 mg/100 mL) Antibody CD127 (mouse monoclonal) Fluidigm Cat#3165008B (1 mg/100 mL) Antibody CXCR5 (rat monoclonal) BD Biosciences Cat#552032 (1 mg/100 mL) Antibody CD27 (mouse monoclonal) Fluidigm Cat#3167006B (1 mg/100 mL) Antibody CD30 (mouse monoclonal) BD Biosciences Cat#555827 (1 mg/100 mL) Antibody CD45RA (mouse monoclonal) Fluidigm Cat#3169008B (1 mg/100 mL) Antibody CD3 (mouse monoclonal) Fluidigm Cat#3170001B (1 mg/100 mL) Antibody CD57 (mouse monoclonal) Biolegend Cat#359602 (1 mg/100 mL) Antibody CD38 (mouse monoclonal) Fluidigm Cat#3172007B (1 mg/100 mL) Antibody CD4 (mouse monoclonal) Fluidigm Cat#3174004B (1 mg/100 mL) Antibody CXCR4 (mouse monoclonal) Fluidigm Cat#3175001B (1 mg/100 mL) Antibody CD25 (mouse monoclonal) Biolegend Cat#356102 (1 mg/100 mL) Antibody PE-CF594 CD27 (mouse monoclonal) BD Biosciences Cat#562297 (1:25–1:200) Antibody BUV737 CD38 (mouse monoclonal) BD Biosciences Cat#564686 (1:25–1:200) Antibody BUV395 CD45RO (mouse monoclonal) BD Biosciences Cat#564291 (1:25–1:200) Antibody FITC CD57 (mouse monoclonal) BD Biosciences Cat#555619 (1:25–1:200) Antibody BV650 CD62L (mouse monoclonal) BD Biosciences Cat#563808 (1:25–1:200) Antibody APC CD69 (mouse monoclonal) Biolegend Cat#310910 (1:25–1:200) Antibody BV421 CD69 (mouse monoclonal) BD Biosciences Cat#562884 (1:25–1:200) Antibody APC CD103 (mouse monoclonal) BD Biosciences Cat#563883 (1:25–1:200) Continued on next page Neidleman et al. eLife 2020;9:e60933.

Techniques: Comparison

Journal: eLife

Article Title: Transcriptional down-regulation of ccr5 in a subset of HIV+ controllers and their family members

doi: 10.7554/eLife.44360

Figure Lengend Snippet:

Article Snippet: Antibody , CD69 mouse Monoclonal Antibody (FN50), FITC , eBioscience , Cat # 11-0699-42; RRID: AB_10853975 , Dilution (1:200).

Techniques: Functional Assay, Recombinant, Derivative Assay, Plasmid Preparation, Generated, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Software