Anti cd45ro apc

Manufactured by BD

Anti-CD45RO-APC is a monoclonal antibody conjugated with the fluorescent dye allophycocyanin (APC) that binds to the CD45RO antigen expressed on the surface of activated and memory T cells. It is used in flow cytometry applications to identify and quantify these cell populations.

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CD45RO-APC (22 citations) Anti-CD45RO (21 citations) APC-labeled anti-CD45RO (3 citations) APC Mouse Anti-Human CD45RO (2 citations)

10 protocols using anti cd45ro apc

Multiparametric Phenotyping of T-Cell Subsets

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Cryopreserved PBMC and cells extracted from the gut biopsy specimens were rested overnight at 37°C before stimulation for 6 h with 40 ng/ml of phorbol myristate acetate (PMA) and 1 µM ionomycin in the presence of 1 µL/mL of brefeldin A (Sigma-Aldrich) to prevent cytokine release. After two washes with RPMI medium containing 10% heat-inactivated human serum (Gemini BioProduct), cells were fixed and permeabilized according to the eBioscience Fix/Perm protocol. The cells were stained with the following antibodies: anti-CD3 PE-Cy7 (Clone:SK7), anti-CD4 APC-Cy7 (Clone:SK3), anti-CD8 PacBlue (Clone:RPA-T8), anti-CD8 PerCP (Clone:SK1), anti-CD27 FITC (Clone:M-T271), anti-CD45RO APC (Clone:), anti-tumor necrosis factor (TNF) APC (Clone: 6401.1111), anti-Ki67 FITC (Clone:MOPC-21) from BD; anti-interferon γ (IFN-γ) PacBlue (Clone:4S.B3) and anti-IL-17 PE (Clone:ebio64Dec17) from eBioscience. Samples were acquired (approximately 200,000 events per sample) and analyzed as described above. In the gut mucosa, T-cells that were CD45RO⁺/CD27⁺ were identified as central memory (CM) and CD45RO⁺/CD27⁻ as effector memory (EM).

Sereti I., Estes J.D., Thompson W.L., Morcock D.R., Fischl M.A., Croughs T., Beq S., Lafaye de Micheaux S., Yao M.D., Ober A., Wilson E.M., Natarajan V., Imamichi H., Boulassel M.R., Lederman M.M, & Routy J.P. (2014). Decreases in Colonic and Systemic Inflammation in Chronic HIV Infection after IL-7 Administration. PLoS Pathogens, 10(1), e1003890.

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Flow Cytometric Analysis of CIK Cells

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CIK cells were resuspended at 2 × 10⁵ cells per 100 μL of phosphate-buffered saline (PBS) and incubated for 30 min at 4 °C with the following anti-human antibodies: anti-CD3-PE-Cy5, anti-CD4-FITC, anti-CD8-PE-CF594, anti-CD25-APC, anti-CD56-PE-Cy7, anti-CD45RO-APC, and anti-CD62L-FITC (all from BD Bioscicence). The cells were analyzed using a CytomicsTM FC500 Flow Cytometer (Beckman Coulter, USA). Data analysis was performed with CXP analysis software (Beckman Coulter, USA).

Zhou Z.Q., Zhao J.J., Pan Q.Z., Chen C.L., Liu Y., Tang Y., Zhu Q., Weng D.S, & Xia J.C. (2019). PD-L1 expression is a predictive biomarker for CIK cell-based immunotherapy in postoperative patients with breast cancer. Journal for Immunotherapy of Cancer, 7, 228.

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Multiparametric Flow Cytometry for Immune Profiling

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The following fluorochrome-conjugated monoclonal antibodies (mAbs) were purchased from commercial vendors and used for surface staining: anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, anti-CD45RO-APC, anti-CCR7-AF700, anti-CD95-PE, anti-CD160-FITC, anti-CD244-FITC, and anti-PD-1-FITC (BD Biosciences, San Jose, CA); anti-CD3-BV605 (Biolegend, San Diego, CA); anti-CD14-Pacific Blue and anti-CD19-Pacific Blue (Life Technologies, Carlsbad, CA); and anti-CD27-PC5 (Beckman Coulter, Indianapolis, IN). The fixable violet amine reactive dye (ViViD; Invitrogen/Molecular Probes, Eugene, OR) was used to eliminate dead cells by flow cytometry. For intracellular cytokine staining, the following mAbs were used: anti-granzyme B (GZMB)-FITC, anti-IL-2-FITC, and anti-IFN-γ-FITC (BD Biosciences).

Hosokawa K., Muranski P., Feng X., Townsley D.M., Liu B., Knickelbein J., Keyvanfar K., Dumitriu B., Ito S., Kajigaya S., Taylor JG V.I., Kaplan M.J., Nussenblatt R.B., Barrett A.J., O’Shea J, & Young N.S. (2016). Memory Stem T Cells in Autoimmune Disease: High Frequency of Circulating CD8+ Memory Stem Cells in Acquired Aplastic Anemia. Journal of immunology (Baltimore, Md. : 1950), 196(4), 1568-1578.

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Multiparametric Flow Cytometry of Tonsillar Cells

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Single cells from tonsil (10 6 ) were stained with Fixable Viability Dye eFluor™ 780 (eBioscience, 65086518, 1:1000) for 20 minutes at 4°C. After wash steps, cells were incubated with a mix of antiCD3BV421 (BD Bio science, 563798, clone SK7, 1:25), antiCD8PerCp (BD Bioscience, 345774, clone SK1, 1:5), antiCD45ROAPC (BD Bioscience, 340438, clone UCHL1, 1:25), and antiCD20PE (Biolegend, 302306, clone 2H7, 1:10) for 30 minutes at 4°C. Next cells were fixed, permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBio science, 00552300) and incubated with antiFOXP3alexa488 (eBioscience, 53477642, clone PCH101, 1:8) 30 minutes at RT. Flow Cytometry was conducted with the FACS Verse (BD Biosciences). Flow cytometry data was analyzed using FlowJo software (v10, Tree Star).

Sultan S., Gorris M.A., Martynova E., Woude L.L.v.d., Buytenhuijs F., Wilpe S.v., Verrijp K., Figdor C.G., Vries I.J.M.d., & Textor J. (2021). ImmuNet: A Segmentation-Free Machine Learning Pipeline for Immune Landscape Phenotyping in Tumors by Muliplex Imaging.

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Isolation and Characterization of TEM Cells

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CD4+ T cells were isolated by negative selection from isolated LPMCs using an Easy Sep Magnet (STEMCELL Technology, Vancouver, Canada) and an Easy Sep Human CD4+ T cell Enrichment kit (STEMCELL Technology). Furthermore, the isolated CD4 positive T cells were stained with anti-CD3-FITC, anti-CD4-PE, anti- CD45RO-APC, anti-CD197 (CCR7) -BV421, and 7ADD-Cell Viability Solution (BD Biosciences), followed by isolation of TEM cells using a FACS Aria II cell sorter (BD Biosciences). Sorting efficiency was consistently over 98%. These TEM cells may include a few regulatory T Cells. However, to keep the number of cells to perform RNA sequencing, we used these samples as TEM cells. DNA and total RNA were extracted from isolated TEM cells using an AllPrep DNA/RNA mini kit (QIAGEN, Hilden, Germany).

Kakuta Y., Ichikawa R., Fuyuno Y., Hirano A., Umeno J., Torisu T., Watanabe K., Asakura A., Nakano T., Izumiyama Y., Okamoto D., Naito T., Moroi R., Kuroha M., Kanazawa Y., Kimura T., Shiga H., Naito T., Esaki M., Kawai Y., Tokunaga K., Nakamura M., Matsumoto T., Nagasaki M., Kinouchi Y., Unno M, & Masamune A. (2020). An Integrated Genomic and Transcriptomic Analysis Reveals Candidates of Susceptibility Genes for Crohn’s Disease in Japanese Populations. Scientific Reports, 10, 10236.

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Multiparametric Flow Cytometry Analysis

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The following fluorochrome-conjugated monoclonal antibodies (mAbs) were purchased from commercial vendors and used for surface staining: anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, anti-CD45RO-APC, anti-CCR7-AF700, anti-CD95-PE, anti-PD-1-FITC and anti-CD69-FITC (BD Biosciences, San Jose, CA); anti-CD3-BV605 (Biolegend, San Diego, CA); anti-CD14-Pacific Blue and anti-CD19-Pacific Blue (Life Technologies, Carlsbad, CA); anti-CD30 Ligand /TNFSF8-AF488 and anti- A20/TNFAIP3- AF488 (Novus Biologicals, Littleton, CO) and anti-CD27-PC5 (Beckman Coulter, Indianapolis, IN). The fixable violet amine reactive dye (ViViD; Invitrogen/Molecular Probes, Eugene, OR) was used to eliminate dead cells by flow cytometry.

Hosokawa K., Kajigaya S., Keyvanfar K., Qiao W., Xie Y., Townsley D.M., Feng X, & Young N.S. (2017). T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways. Journal of immunology (Baltimore, Md. : 1950), 199(2), 477-488.

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Flow Cytometry Immunophenotyping Panel

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The following antibodies were used for flow cytometry: anti Integrin β7 PE (clone FIB504), anti CD45RO APC and anti CD45RO BV421 (clone UCHL1), anti CD62L PE (clone DREG-56), anti CD69 PE (clone FN50), anti CCR5 APC (clone 2D7) and anti Ki-67 FITC (clone MOPC-21) (BD Pharmingen, San Diego, CA). For intracellular Ki-67 staining, BD Perm Wash Kit and company protocol were utilized (BD Pharmingen, San Diego, CA). The anti-α4β7 mAb heterodimer (clone Act-1) was gifted by Dr. Aftab Ansari. The Synagis antibody was gifted from Dr. Barton Haynes and used as a control in several experiments where designated. Data were collected on a FACSCanto II (BD Biosciences, San Diego, CA) and were analyzed using FlowJo and GraphPad Prism software.

Nawaz F., Goes L.R., Ray J.C., Olowojesiku R., Sajani A., Ansari A.A., Perrone I., Hiatt J., Van Ryk D., Wei D., Waliszewski M., Soares M.A., Jelicic K., Connors M., Migueles S.A., Martinelli E., Villinger F., Cicala C., Fauci A.S, & Arthos J. (2018). MAdCAM costimulation through Integrin-α4β7 promotes HIV replication. Mucosal immunology, 11(5), 1342-1351.

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Flow Cytometry Immunophenotyping Panel

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Expression and Phenotyping of CD19-CARs on T Cells

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Expression of the various CD19-CARs on T cells was detected using either biotinylated anti-mouse FMC63 scFv monoclonal antibody (Biowan) followed by staining with streptavidin-phycoerythrin (PE) (BD Biosciences), or with an allophycocyanin (APC)-labeled anti-mouse FMC63 scFv monoclonal antibody (Biowan). 1 × 10⁶ cells were stained for cell surface markers to analyze T cell differentiation and exhaust status. The following antibodies were used: anti-CD3-FITC (BD Biosciences), anti-human CD3-APC (BD Biosciences), anti-CCR7-BV421 (BD Biosciences), anti-CD 45RO-APC (BD Biosciences), anti-CD4-BB515, anti-CD8-BV510 (BD Biosciences), anti-CD45RA-PE- Cy7 (BD Biosciences), anti-CD62L-PE (BD Biosciences), anti-PD-1-APC (BD Biosciences), anti-TIM-3APC (BD Biosciences), and anti-LAG-3-APC (BD Biosciences). Samples were analyzed on either FACSCanto II flow cytometers (BD Biosciences) or Novocyte3110 (AECE).

Sun M., Xu P., Wang E., Zhou M., Xu T., Wang J., Wang Q., Wang B., Lu K., Wang C, & Chen B. (2021). Novel two-chain structure utilizing KIRS2/DAP12 domain improves the safety and efficacy of CAR-T cells in adults with r/r B-ALL. Molecular Therapy Oncolytics, 23, 96-106.

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Phenotyping of Monocyte-Derived DCs and T Cells

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Cells were harvested and washed twice with PBS containing 1% BSA (flow cytometry buffer). The following antibody cocktail was used to phenotype moDCs: anti-CCR7-APC (clone G043H7, BioLegend), anti-CD40-BV605 (clone 5C3, BD), anti-CD86-FITC (clone FUN-1, BD Pharmingen), anti-HLA-DR-PE-CY7 (clone G46-6, BD Pharmingen), and anti-HLA-A2-BV421 (clone BB7.2, BioLegend). Cells were also stained with 7-AAD (BioLegend) to discriminate live from dead cells. T cell phenotyping was performed using the following antibodies: anti-CD8-BV421 (clone RPA-T8, BioLegend), anti-CD45RA-PE (clone HI100, BD), anti-CD45RO-APC (clone UCHL1, BD) and MHC dextramer Mel-A-PE or -APC (HLA-A∗0201/ELAGIGILTV, WB2162, Immudex) for CD8⁺ T cells and anti-CD4-FITC (clone OKT4, BioLegend), anti-CD69-PerCP-CY5.5 (clone FN50, BioLegend) and anti-OX40-APC (clone ACT35, BioLegend) for CD4⁺ T cells. T cells were also stained with 7-AAD (BioLegend) to discriminate live from dead cells. Cells were acquired on the LSR Fortessa flow cytometer and analyzed with FlowJo software, version 10.0.

de Mey W., De Schrijver P., Autaers D., Pfitzer L., Fant B., Locy H., Esprit A., Lybaert L., Bogaert C., Verdonck M., Thielemans K., Breckpot K, & Franceschini L. (2022). A synthetic DNA template for fast manufacturing of versatile single epitope mRNA. Molecular Therapy. Nucleic Acids, 29, 943-954.

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