Cd69 clone fn50

Manufactured by BD

Sourced in United Kingdom

CD69, also known as CLEC2A, is a cell surface glycoprotein that serves as an early activation marker on various immune cells, including T cells, B cells, and natural killer cells. The FN50 clone is a commonly used monoclonal antibody for the detection and quantification of CD69 expression. This product is intended for research use only and its core function is to facilitate the identification and analysis of CD69-expressing cell populations.

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CD69 (FN50, (7 citations) CD69-PE (clone FN50), (6 citations) Clone FN50 (4 citations) CD69-PerCP-Cy5–5 (clone FN50, (3 citations) Anti CD69 PE (clone FN50), (2 citations) CD69-PE.Cy7 (clone FN50; (2 citations) Anti-CD69 (clone FN50, (2 citations)

7 protocols using cd69 clone fn50

CFSE-labeled Cell Cytotoxicity Assay

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The day prior to the assay, 3×10⁴ JEG-3 cells were labeled with 1 µM of CFSE (CellTrace, Thermo Fisher) and seeded in flat-bottom 96-well microplates. On the day of the assay, CAR-T cells were added at various E:T ratios to either the plated CFSE-labeled JEG-3 cells or 3×10⁴ CFSE-labeled K562/K562-HLA-G1 cells. After 24 hours incubation, medium was collected and cells recovered, washed and labeled with antibodies against CD4 (clone RPA-T4, Biolegend), CD8 (clone RPA-T8, Biolegend), CD19 (HIB-19, Biolegend), CD25 (clone M-A251, BD Bioscience), CD69 (clone FN-50, BD Bioscience), PD-1 (EH12.2h7, Biolegend) and a viability dye (Invitrogen). For degranulation assays, co-cultures were set-up at an E:T ratio of 10:1. Anti-CD107a (clone eBioH4A3, Biolegend) was added at the start of the experiment, GolgiStop (BD Bioscience) was added after 1 hour. Five hours after the beginning of the assay, cells were collected and labeled with antibodies directed against CD4, CD8, CD19 and a viability dye. Acquisition was performed with a fluorescence-activated cell sorting (FACS) Attune (Thermo Fisher), and results were analyzed with FlowJo software.

Anna F., Bole-Richard E., LeMaoult J., Escande M., Lecomte M., Certoux J.M., Souque P., Garnache F., Adotevi O., Langlade-Demoyen P., Loustau M, & Caumartin J. (2021). First immunotherapeutic CAR-T cells against the immune checkpoint protein HLA-G. Journal for Immunotherapy of Cancer, 9(3), e001998.

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Antigen-Specific T Cell Activation Analysis

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Cryopreserved PBMCs were thawed and 10⁶ live cells were plated out in 96 well U-bottom plates. Spike protein peptide pool (GenScript, New Jersey, USA), or Chimpanzee Adenovirus Y25 hexon protein peptide pool (JPT Peptide Technologies, Berlin, Germany) was added to each well at 1 μg/mL and cells incubated for 20–24 h at 37°C. For surface staining of AIM markers, cells were incubated in 1 μg/mL human Fc block (BD Biosciences) and fixable viability solution 780 (BD Biosciences) in PBS for 15 min and washed in PBS. An antibody cocktail containing antibodies against CD3 (clone UCHT1, BD Biosciences), CD4 (clone M-T477, BD Biosciences), CD8 (G42-8, BD Biosciences), CXCR5 (J252D4, BD Biosciences), CD38 (HIT2, BD Biosciences), PD-1 (EHI2.1, BD Biosciences), CD69 (clone FN50, BD Biosciences), CD137 (clone 4B4-1, Biolegend), OX40 (clone Ber-ACT35, Biolegend), CCR7 (clone 2-L1-A, BD Biosciences), and CD45RA (clone HI100, BD Biosciences)were added directly to cells and incubated for a further 30 min at 4°C. Following surface staining, cells were washed twice in PBS. All samples were acquired on a BD FACS Symphony and analyzed using FlowJO software v18 (FlowJo, Ashland, USA). The gating strategy for AIM⁺ T cells is shown in (Methods S3). Data were imported into R v4.2 and visualized with the ggplot2 v3.3.6 package.

Ryan F.J., Norton T.S., McCafferty C., Blake S.J., Stevens N.E., James J., Eden G.L., Tee Y.C., Benson S.C., Masavuli M.G., Yeow A.E., Abayasingam A., Agapiou D., Stevens H., Zecha J., Messina N.L., Curtis N., Ignjatovic V., Monagle P., Tran H., McFadyen J.D., Bull R.A., Grubor-Bauk B., Lynn M.A., Botten R., Barry S.E, & Lynn D.J. (2023). A systems immunology study comparing innate and adaptive immune responses in adults to COVID-19 mRNA and adenovirus vectored vaccines. Cell Reports Medicine, 4(3), 100971.

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CD25 and CD69 Expression in Activated T Cells

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A half million resting CD4 T cells were cultured for 5 days and stimulated with anti-CD3/CD28 beads (4 beads per cell) for 24 h. The cells were stained with phycoerythrin (PE)-labeled monoclonal antibody against human CD25 (clone M-A251) or CD69 (clone FN50) (BD Biosciences, San Jose, CA). The cells were stained on ice in PBS plus 0.1% BSA for 30 min, washed with cold PBS-0.5% BSA, and then analyzed on a FACSCalibur (BD Biosciences, San Jose, CA).

Yi F., Guo J., Dabbagh D., Spear M., He S., Kehn-Hall K., Fontenot J., Yin Y., Bibian M., Park C.M., Zheng K., Park H.J., Soloveva V., Gharaibeh D., Retterer C., Zamani R., Pitt M.L., Naughton J., Jiang Y., Shang H., Hakami R.M., Ling B., Young J.A., Bavari S., Xu X., Feng Y, & Wu Y. (2017). Discovery of Novel Small-Molecule Inhibitors of LIM Domain Kinase for Inhibiting HIV-1. Journal of Virology, 91(13), e02418-16.

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T Cell Signaling Pathway Analysis

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Antibodies used for T cell stimulation included DimerX I recombinant HLA-A2 Ig (BD Biosciences), CD3 (clone OKT3, Biolegend), and CD28 (clone CD28.2, Biolegend). Western blot antibodies included PTPN22 (clone D6D1H, Cell Signaling Technology), Lck (Cell Signaling Technology), Zap-70 (Cell Signaling Technology), α-tubulin (clone TU-02, Santa Cruz), anti-rabbit Ig (Life Technologies) and anti-mouse Ig (LI-COR). For IP-FCM, the Akt capture antibody used was clone SKB1 (Merck Millipore), total Akt was detected using clone 53G (Cell Signaling Technology), and phospho Akt S473 was detected using clone D9E (Cell Signaling). Flow cytometry antibodies included CD69 (clone FN50, BD Biosciences), Il-2 (clone MQ1-17H12, Biolegend), pSrc family Y416 (Cell Signaling Technology), Lck pY505 (Cell Signaling Technology), Zap-70 pY493 (Cell Signaling Technology), Erk 1/2 pY204 (clone 197G2, Cell Signaling Technology), NFAT (clone D43B1, Cell Signaling Technology), cFos (clone 2G2, Novus Biologicals), an anti-rabbit secondary (Invitrogen), and an anti-mouse secondary (Life Technologies).

Bray C., Wright D., Haupt S., Thomas S., Stauss H, & Zamoyska R. (2018). Crispr/Cas Mediated Deletion of PTPN22 in Jurkat T Cells Enhances TCR Signaling and Production of IL-2. Frontiers in Immunology, 9, 2595.

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Multiparametric Flow Cytometry Analysis

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Following BFA treatment for the final 4 h, cells were resuspended in 1:1000 dilution of Fixable Viability Stain (Life Technologies, Gloucester, UK) for LIVE/DEAD cell determination. Cells were subsequently resuspended in PBS with 1% BSA (Sigma-Aldrich) and treated with Fcγ block (Life Technologies). For extracellular cell staining, cells were labelled with fluorochrome-conjugated Abs against CD3 (clone OKT3; Life Technologies), CD69 (clone FN50; BD Biosciences, Wokingham, UK), CD86 (clone IT2.2; Biolegend), DC-SIGN (clone eB-h209; Life Technologies) and CD107a (clone H4A3; Biolegend). Cells were then fixed and permeabilized using the FIX and PERM Kit (Life Technologies) before intracellular staining with fluorochrome-conjugated Abs against IFNγ (clone 4S.B3; Biolegend), IL-17A (clone eBIO64 DEC17; Life Technologies), Granzyme B (clone GB11; Biolegend), perforin (clone B-D48; Biolegend), granulysin (clone DH2; Biolegend), and granzyme A (clone CB9; Biolegend). For apoptosis analysis, cells were stained with Abs against Annexin V (Biolegend) and with propidium iodide (PI) (Biolegend) before immediate acquisition. Flow cytometric data were acquired with a BD LSRFortessa (BD Biosciences) and analysed using FlowJo v10.6.2 (Treestar, Ashland, OR, USA). Fluorescence minus one controls were used for the setting of gates.

Cooper A.J., Clegg J., Cassidy F.C., Hogan A.E, & McLoughlin R.M. (2022). Human MAIT Cells Respond to Staphylococcus aureus with Enhanced Anti-Bacterial Activity. Microorganisms, 10(1), 148.

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CD1a-restricted BK6 Jurkat Cell Activation

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CD1a-restricted BK6 Jurkat cells were generated as previously described³⁷. 1×10⁴ C1R CD1a cells (wild type or mutant) were mixed with 2×10⁴ BK6 Jurkat cells and cultured overnight. Following overnight culture, cells were stained with CD3 (clone UCHT1, BD biosciences), CD69 (clone FN50, BD biosciences) and CD19 (clone H1B19, Biolegend) and activation of CD3⁺ BK6 Jurkat cells assessed by CD69 up-regulation. Cells were analysed using a LSR Fortessa (BD sciences) and data processed using FlowJo software (Tree Star Inc.).

Birkinshaw R.W., Pellicci D.G., Cheng T.Y., Keller A.N., Sandoval-Romero M., Gras S., de Jong A., Uldrich A.P., Moody D.B., Godfrey D.I, & Rossjohn J. (2015). αβ T-cell receptor recognition of CD1a presenting self-lipid antigens. Nature immunology, 16(3), 258-266.

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Multiparameter Flow Cytometry of Immune Cells

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PBMCs, cervical cells and rectal cells were stained with pre-determined concentrations of antibodies directed against CD3 (clone SK7) (eBioscience), CD4 (clone SK3) (BD Horizon), CCR5 (clone 2D7) (BD Horizon), CD69 (clone FN50) (BD Pharmingen), CD49d (clone 9F10) (eBioscience), and β7 (clone FIB504) (BD Pharmingen). Dead cells were marked using LIVE/DEAD Far Red Cell Stain Kit (Invitrogen). Some samples were also stained with antibodies against CD103 (clone Ber-ACT8) (BioLegend). An LSRII flow cytometer driven by the DiVa software package (BD Biosciences) was used to acquire the samples. Analysis was performed on FlowJo v10.1 software (FlowJo, LLC, USA).

Perciani C.T., Jaoko W., Farah B., Ostrowski M.A., Anzala O, & MacDonald K.S. (2018). αEβ7, α4β7 and α4β1 integrin contributions to T cell distribution in blood, cervix and rectal tissues: Potential implications for HIV transmission. PLoS ONE, 13(2), e0192482.

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