Anti cd69

Manufactured by BD

Sourced in United States

Anti-CD69 is a laboratory reagent used for the detection and analysis of CD69 expression on cells. CD69 is a cell surface glycoprotein that serves as an early activation marker for various immune cell types. The Anti-CD69 reagent can be used in flow cytometry and other immunoassays to identify and quantify CD69-positive cells.

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Lab products found in correlation

Anti cd4, by BD (15 mentions) Anti cd8, by BD (13 mentions) Anti cd11b, by BD (12 mentions) Anti cd3, by BD (10 mentions) Anti cd45, by BD (6 mentions) Anti ifn γ, by BD (5 mentions) Anti cd62l, by BD (5 mentions) Anti cd44, by BD (5 mentions) Anti hla dr, by BD (5 mentions) Anti cd11c, by BD (4 mentions) Anti gr 1, by BD (3 mentions) Facsaria 2 cell sorter, by BD (3 mentions) Anti tnf α, by BD (3 mentions) Anti f4 80, by Thermo Fisher Scientific (3 mentions) Anti nkp46, by BioLegend (3 mentions) Anti cd38, by BD (3 mentions) Anti b220, by BD (3 mentions) Anti ly6g, by BD (3 mentions) Anti nk1, by BD (3 mentions) Anti cd3, by BioLegend (3 mentions)

Close spelling variants of this product

Anti-CD69-PE (34 citations) Anti-CD69-PE/Cy7 (11 citations) Anti-CD69 APC (10 citations) PE-conjugated anti-CD69 (10 citations) Anti-CD69 (H1.2F3, (7 citations) Anti-CD69 APC-Cy7 (5 citations) PE mouse anti-human CD69 (4 citations) Anti-CD69–FITC (clone L78; (3 citations) Phycoerythrin‐conjugated anti‐CD69 (3 citations) PE-conjugated mouse anti-human CD69 antibody (3 citations)

38 protocols using anti cd69

Multiparametric Flow Cytometry Analysis of Immune Cell Subsets

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Surface and intracellular staining were performed as previously described [28 (link)]. Single immune cell populations from the spleen, lymph nodes or tumors were separated with a BD FACSAria II Cell Sorter. Flow cytometric analyses were performed with Flowjo (Tree Star). The following antibodies were used for cell staining: anti-CD3 (clone 145-2C11), anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD49b (clone DX5), anti-CD11b(clone M1/70), anti-CD11c (clone HL3), anti-CD19 (clone 1D3), anti-CD25 (clone PC61), anti-CD69 (clone H1.2F3), anti-CD62L (MEL-14), anti-CD44 (clone IM7), anti-Foxp3 (clone FJK-16s), anti-Granzyme B (clone GB11), anti-CCR4 (clone 2G12), anti-CCR5 (clone HM-CCR5), anti-CXCR3 (clone CXCR3-173), NK1.1(clone PK136), anti-F4/80 (clone BM8), anti-Gr-1 (clone RB6-8C5), anti-interferon-γ (IFN-γ, clone XMG1.2), and anti-NK1.1 (clone PK136), CD4 blocking mAb (clone GK1.5), and CD8 blocking mAb (clone 53-6.7).
For detection of phosphorylated S6 proteins, cells from LNs cultured with PMA (10ng/ml) and Ionomycin (500ng/ml) at designated times were immediately fixed with phosflow Lyse/Fix buffer (BD Biosciences) and permeabilized by Phosflow Perm buffer (BD Biosciences). Cells were stained with the Alex488 conjugated antibody for S6P (Ser235,236) (D57.2. 2E; Cell Signaling Technology)

Rao E., Zhang Y., Zhu G., Hao J., Persson X.M., Egilmez N.K., Suttles J, & Li B. (2015). Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death. Oncotarget, 6(10), 7944-7958.

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Multiparametric Flow Cytometry Analysis

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FITC-labelled anti-IA/IE, PE-labelled anti-Vβ11, anti-TNFα, anti-CD45.2, anti-CD40, APC-conjugated anti-CD80, anti-IFNγ, anti-CD69, PeCy7-labelled anti-CD11c, PercypCy5.5-labeled anti-CD44, anti-IL-2 and APC-Cy7-conjugated anti-CD62L were obtained from BD Pharmingen (San Diego, CA, USA). PE- labelled anti-CD86, PE-efluor 610-labelled anti-CD25 and Pacific blue conjugated-Annexin V were from Biolegend (San Diego, CA, USA). Alexa 405-conjugated anti-CD4 was obtained from CALTAG lab (Buckingham, UK). APC-conjugated anti-IA/IE and PE-labelled anti-CCR7 were obtained from eBioscience (San Diego, CA, USA). All stainings were performed in presence of mouse Fc Block (BD Biosciences). Dead cells were stained using the Live/Death detection kit with a near-infrared dye (Invitrogen). The samples were analysed using CyAn ADP Analyser (Beckman Coulter, Brea, CA, USA) and the data were analysed using FlowJo version 9.2 (Tree Star inc, Ashland, Oregon, USA).

Nasi A., Bollampalli V.P., Sun M., Chen Y., Amu S., Nylén S., Eidsmo L., Rothfuchs A.G, & Réthi B. (2017). Immunogenicity is preferentially induced in sparse dendritic cell cultures. Scientific Reports, 7, 43989.

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Multiparametric PBMC Phenotyping

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PBMCs were isolated by Histopaque-1077 (Sigma Aldrich, USA) density gradient centrifugation and, after gating for CD3 positivity, were stained with anti-CD4 PE, anti-CD25FITC (BD Biosciences, USA), anti-CD69 and anti-FoxP3 for FACS fluorescence cytometry. After gating for Lin-negativity, the isolated PBMCs were also stained for the myeloid dendritic markers CD11c and CD86PE (eBioscience, USA). The data were analyzed with a Beckman Coulter flow cytometer.

Petrou P., Kassis I., Ginzberg A., Halimi M., Yaghmour N., Abramsky O, & Karussis D. (2021). Long-Term Clinical and Immunological Effects of Repeated Mesenchymal Stem Cell Injections in Patients With Progressive Forms of Multiple Sclerosis. Frontiers in Neurology, 12, 639315.

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Multiparameter Flow Cytometry of Brain Immune Cells

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Brain mononuclear cells extracted with Percoll were stained with anti-NKp46 (BioLegend, catalog no. 137618), anti-CD3 (BD Biosciences, catalog no. 553066), anti-CD45 (BD Biosciences, catalog no. 559864), anti-CD11b (BD Biosciences, catalog no. 552850), anti-F4/80 (Thermo Fisher Scientific, catalog no. 12-4801-82), anti-CD4 (BD Biosciences, catalog no. 557308), and anti-CD8 (BD Biosciences, catalog no. 553030) antibodies for flow cytometric analysis of immune cell infiltration into the brain. Anti-IFN-γ (BD Biosciences, catalog no. 563773), anti-CD69 (BD Biosciences, catalog no. 564683), and anti-GZMB (Thermo Fisher Scientific, catalog no. 48-8898-82) antibodies were used to measure immune cell activation. The flow cytometric assessments were performed with four or more independent animals. Anti-human CD191 (CCR1, BioLegend, catalog no. 362907) and anti-human CD195 (CCR5, BioLegend, catalog no. 313707) antibodies were used to detect their expressions on human NK cells, macrophages and T cells. All flow cytometry data were collected using a Fortessa X-20 flow cytometer. Cell sorting was performed using a FACS Aria II cell sorter (BD Biosciences). The gating strategy is shown in Supplementary Fig. 3. BD FACSDiva and FlowJo 7.6 & 10.0 were used to collect and analyze the flow cytometric data, respectively.

Zhu Q.Y., Headrick J.P, & Berne R.M. (1991). Transmural distribution of extracellular purines in isolated guinea pig heart.. Proceedings of the National Academy of Sciences of the United States of America, 88(2), 657-660.

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Comprehensive Lymphocyte Phenotyping

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Lymphocytes isolated from peripheral blood, LN, jejunum, and rectum were quantified for their activation (CD38+, CD69+, and HLADR+), proliferation (Ki67+), and naïve/memory phenotype (CD28+ and CD95+). Cells were first stained with live/dead stain (Thermo Fisher Scientific, Eugene, OR, USA). After washing, cells were further stained with cocktail of monoclonal antibodies (mAbs) including anti-CD3, anti-CD4, anti-CD8, anti-CD28, anti-CD95, anti-CD38, anti-CD69, and anti-HLADR antibodies (BD Biosciences, San Jose, CA, USA; Table S2) as reported earlier. To detect proliferating Ki67+ cells, intracellular staining protocol was performed using BD Fix/Perm solutions as described earlier [26 (link)]. Cells were fixed with 1x BD stabilizing and fixative buffer. At least 20,000 events were collected by gating on lymphocytes from each sample, and the data were analyzed using FlowJo software, version 10.7.0. (Becton, Dickinson & Company, Franklin Lakes, NJ, USA).

Pahar B., Gray W., Fahlberg M., Grasperge B., Hunter M., Das A., Mabee C., Aye P.P., Schiro F., Hensley K., Ratnayake A., Goff K., LaBranche C., Shen X., Tomaras G.D., DeMarco C.T., Montefiori D., Kissinger P., Marx P.A, & Traina-Dorge V. (2022). Recombinant Simian Varicella Virus-Simian Immunodeficiency Virus Vaccine Induces T and B Cell Functions and Provides Partial Protection against Repeated Mucosal SIV Challenges in Rhesus Macaques. Viruses, 14(12), 2819.

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Multiparameter Flow Cytometry Analysis

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Spleen cells were isolated as described above and placed in ice-cold PBS supplemented with 5% FCS and 0.1% sodium azide. Staining was performed as previously described [11 (link)]. The following fluorochrome-conjugated monoclonal antibodies were used: anti-CD4 [H129.19]; anti-CD8 [53-6.7]; anti-CD19 [MB19-1]; anti-CD25 [7D4]; anti-CD44 [IM7]; anti-CD69 [H1.2F3]; anti-Gr-1/Ly6C/Ly6G [RB6-8C5]; anti-CD45RB [16A]; anti-CD62L [MEL-14]; anti-CD11b [M1/70] (BD Pharmingen, San Diego, CA); and anti-CD11c [HL3]—(Serotech, Raleigh, NC) anti-F4/80 [CI:A3-1] and anti-GITR [DTA-1] (eBioscience, San Diego, CA). After staining, the cells were fixed with 1% paraformaldehyde in PBS and analyzed using a FACSCanto (BD Biosciences, San Jose, CA), 50,000 events/sample recorded. Data were processed using FlowJo software (FlowJo LLC, Ashland, OR). Cell numbers were calculated using the percentage obtained by FACS analysis and the total numbers of leukocytes counted in a hemocytometer.

Canavaci A.M., Sorgi C.A., Martins V.P., Morais F.R., de Sousa É.V., Trindade B.C., Cunha F.Q., Rossi M.A., Aronoff D.M., Faccioli L.H, & Nomizo A. (2014). The Acute Phase of Trypanosoma cruzi Infection Is Attenuated in 5-Lipoxygenase-Deficient Mice. Mediators of Inflammation, 2014, 893634.

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Immune Cell Profiling in Tumor, Liver, and Kidney

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Mononuclear cells in the tumor site, liver, and kidney were extracted with Percoll and stained with anti-NKp46 (Biolegend, cat#137618), anti-CD3 (BD, cat#553066), anti-CD45 (BD, cat#559864), anti-CD11b (BD, cat#552850), anti-F4/80 (Thermo, cat# 12–4801-82), anti-CD4 (BD, cat# 557308) and anti-CD8 (BD, cat# 553030), anti-CD86 (BD, cat#740900), anti-CD206 (Biolegend, cat#141734), anti-CD69 (BD, cat#564683), anti-IFNγ (BD, cat#563773), anti-granzyme B (Thermo Fisher, cat#48–8898-82) antibodies for flow cytometric analysis of immune cell infiltration and activation. H-2K(b) herpes simplex virus type 1 glycoprotein B tetramer was provided by the NIH Tetramer Core Facility. The flow cytometric assessments of murine immune cells were performed with at least 3 independent animals. All flow cytometry data were collected using the Fortessa X-20 flow cytometer except cell sorting experiments that were performed using FACS Aria II cell sorter (BD Biosciences).

Tian L., Xu B., Teng K.Y., Song M., Zhu Z., Chen Y., Wang J., Zhang J., Feng M., Kaur B., Rodriguez L., Caligiuri M.A, & Yu J. (2021). Targeting Fc receptor-mediated effects and the “don’t eat me” signal with an oncolytic virus expressing an anti-CD47 antibody to treat metastatic ovarian cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 28(1), 201-214.

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Multi-parameter Flow Cytometric Analysis of Immune Cell Populations

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Single-cell suspensions were resuspended in BD stain buffer (BD Bioscience) for 15 min prior to staining with specific antibodies. Antibodies against cell markers: anti-CD45, anti-CD3, anti-CD4, anti-CD8a, anti-CD11b, anti-F4/80, anti-Ly6G, anti-Ly6C, anti-NKp46(CD335), anti-B220, and anti-CD69, were purchased from BD Bioscience and BioLegend; anti-OX40L and anti-41BBL from Miltenyi Biotec (detailed in Supplementary table 4). Samples were mixed with FVS700 (1/7000) and data were acquired on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star). ROS generation in cultured neutrophils was determined using either DCFDA/H2DCFDA-Cellular ROS Assay Kit (ab113851) following the manufacturer’s protocol or a dihydroethidium (DHE) fluorescent probe. Cells were incubated with DHE (10 μM) in HBSS containing 1.5 mM CaCl₂ and 1 mM MgCl₂ for 30 min at 37 °C and analyzed by flow cytometry. LacZ activity was determined by flow cytometry using Fluorescein di[β-D-galactopyranoside (Sigma, F2756) as described elsewhere^{94 (link)}.

Uyanik B., Goloudina A.R., Akbarali A., Grigorash B.B., Petukhov A.V., Singhal S., Eruslanov E., Chaloyard J., Lagorgette L., Hadi T., Baidyuk E.V., Sakai H., Tessarollo L., Ryffel B., Mazur S.J., Lirussi F., Garrido C., Appella E, & Demidov O.N. (2021). Inhibition of the DNA damage response phosphatase PPM1D reprograms neutrophils to enhance anti-tumor immune responses. Nature Communications, 12, 3622.

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Apoptosis and Activated T Cell Markers

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For detection of apoptotic cells, cells were stained as above. For detection of CD69, CD44, and CD62L, LN cells isolated from naïve mice or mice on day 8 after immunization were stimulated with OVA protein (0, 1, 10, 100 μg/ml) or anti-CD3ε antibody (1 μg/ml) and stained with anti-CD69 (PharMingen), anti-CD44 (TONBO Biosciences), and anti-CD62L (BD Biosciences) antibodies on day 0 or 2. For detection of OTII-TCR-Tg-positive cells, cells were stained with anti-TCR Vα2-FITC antibody (clone B20.1, BioLegend). Stained cells were analyzed with BD FACSVerse^TM (BD Biosciences) for detection of respective surface molecules.

Tong H., Miyake Y., Mi-ichi F., Iwakura Y., Hara H, & Yoshida H. (2018). Apaf1 plays a negative regulatory role in T cell responses by suppressing activation of antigen-stimulated T cells. PLoS ONE, 13(3), e0195119.

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Phenotyping Immune Cells by Flow Cytometry

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Conjugated antibodies purchased from BD Bioscience were anti-CD8, anti-CD4, anti-CD69, anti-CD62L, anti-CD44, anti-CD122, anti-CD107a, anti-IFN-γ, anti-TNF-α and Ki-67. Granzyme B antibody was purchased from Life technologies. BrdU kit was purchased from BD biosciences. HSV gB_498–505 peptide (SSIEFARL) was supplied by Genscript. PE-Kb-gB tetramer was kindly provided by NIH tetramer core facility, Emory University, Atlanta, GA. The antibody-stained cells were acquired with a FACS Calibur (BD Biosciences) and the data were analyzed using the FlowJo software (Tree Star, OR).

Rajasagi N.K, & Rouse B.T. (2016). IL-2 complex treatment amplifies CD8+ T cell mediated immunity following herpes simplex virus-1 infection. Microbes and infection, 18(12), 735-746.

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