Cd45ro fitc

Manufactured by BioLegend

CD45RO-FITC is a fluorescently labeled antibody that recognizes the CD45RO isoform of the CD45 protein, which is expressed on the surface of memory T cells. The FITC (fluorescein isothiocyanate) fluorescent dye is conjugated to the antibody, allowing for the identification and analysis of CD45RO-positive cells using flow cytometry.

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Anti-CD45RO FITC (2 citations)

5 protocols using cd45ro fitc

Multicolor Flow Cytometry for T and B Cell Subsets

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PBMC were isolated from peripheral blood by Ficoll gradient and frozen for batched analysis. We designed six multicolor flow cytometry panels to quantify 60 T cell subsets along with two B cell subsets, and calculated the CD4⁺/CD8⁺ T cell ratio (Supplementary Table 1). The following fluorochrome-conjugated anti-human antibodies were used from BD Biosciences (San Jose, CA): CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CCR4-PE, CD27-PE, CD28-BV421, CD138-BV421, CCR6-BV421, CXCR3-PE, CCR7-A700, IL-17-BV786, IFN-γ-PE-Cy7, iso IgG1k-FITC, iso IgG1k-PE-Cy7, iso IgG2bk-APC, iso IgG1k-APC-Cy7, and iso IgG1k-BV510; from Biolegend (San Diego, CA): CD127-FITC, CD27-APC, CD57-PerCp-Cy5.5, CD19-BV510, PD-1-APC-Cy7, CXCR5-FITC, and TNFα-FITC; from eBioscience (San Diego, CA): CD4-PE-Cy7, and IL-2-PE; from Miltenyi Biotec (San Diego, CA): CD25-APC and KLRG1-PE; from Beckman Coulter (Brea, CA): CD38-PE-Cy7.

Hartzell S., Bin S., Cantarelli C., Haverly M., Manrique J., Angeletti A., Manna G.L., Murphy B., Zhang W., Levitsky J., Gallon L., Yu S.M, & Cravedi P. (2020). Kidney Failure Associates With T Cell Exhaustion and Imbalanced Follicular Helper T Cells. Frontiers in Immunology, 11, 583702.

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Phenotypic Profiling of T Cells

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For baseline T cell frequencies 0.5(10)⁶ cells from baseline uninfected total lymphocyte samples were stained and analyzed as above with phenotype antibody panel as follows: CD95-APC (2μl, Biolegend 305611), CCR7-PE (2μl, BD 566742), CD28-PE Cy7 (2μl, Biolegend 302925), CD45RO-FITC (3μl, Biolegend 304204), CD45RA-PerCP Cy5.5 (2μl, Biolegend 304121), CD4-APC H7 (2μl, BD 560158), CD19-V510 (3μl, BD 562953), CD8-V450 (2.5μl, BD 561426).

Alomari N, & Totonchy J. (2022). IL-21 signaling promotes the establishment of KSHV infection in human tonsil lymphocytes by increasing differentiation and targeting of plasma cells. Frontiers in Immunology, 13, 1010274.

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Multicolor Flow Cytometry of PBMC

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We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4⁺, CD8⁺, and CD19⁺. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 10⁶ events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo^® software.

Tawhari I., Hallak P., Bin S., Yamani F., Safar-Boueri M., Irshad A., Leventhal J., Ansari M.J., Cravedi P, & Gallon L. (2022). Early calcineurin-inhibitor to belatacept conversion in steroid-free kidney transplant recipients. Frontiers in Immunology, 13, 1096881.

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Flow Cytometric Analysis of Activated Cells

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Cells were harvested after stimulation and viability of cells post activation was measured using the LIVE/DEAD^™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Surface staining of antigen was performed at 4°C for 20 minutes. For intracellular antigens, cells were fixed and permeabilized using the FOXP3/Transcription factor staining kit (eBioscience), and incubated with the appropriate antibody for 1 hour at 4°C. Acquisition of cells was performed using the BD FACSCantoTM II Flow Cytometer and subsequent analysis was performed using FlowJo (v9 or v10). The clone and dilution of antibodies used for flow cytometry were as follows: CD4-Pacific Blue (Biolegend, OKT4, 1:200), CD69-APC (Biolegend, FN50, 1:200), CD45RA-APC (Biolegend, HI100, 1:200), CD45RO-FITC (Biolegend, UCHL1, 1:200), DEFA1 (R&D Systems, 2μg/ml), anti-sheep IgG-NL557 (R&D Systems, 1:500). IFNγ expression was analyzed using the IFN-γ Secretion Assay (PE, Miltenyi Biotec) following manufacturer’s instructions.

Bibby J.A., Agarwal D., Freiwald T., Kunz N., Merle N.S., West E.E., Singh P., Larochelle A., Chinian F., Mukherjee S., Afzali B., Kemper C, & Zhang N.R. (2022). Systematic Single Cell Pathway Analysis (SCPA) to characterize early T cell activation. Cell reports, 41(8), 111697.

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Viability and Surface Antigen Analysis

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Cells were harvested after stimulation and viability of cells post activation was measured using the LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Surface staining of antigen was performed at 4 C for 20 minutes. For intracellular antigens, cells were fixed and permeabilized using the FOXP3/Transcription factor staining kit (eBioscience), and incubated with the appropriate antibody for 1 hour at 4 C. Acquisition of cells was performed using the BD FACSCantoTM II Flow Cytometer and subsequent analysis was performed using FlowJo (v9 or v10).
The clone and dilution of antibodies used for flow cytometry were as follows: CD4-Pacific Blue (Biolegend, OKT4, 1:200), CD69-APC (Biolegend, FN50, 1:200), CD45RA-APC (Biolegend, HI100, 1:200), CD45RO-FITC (Biolegend, UCHL1, 1:200), DEFA1 (R&D Systems, 2 g/ml), anti-sheep IgG-NL557 (R&D Systems, 1:500). IFN expression was analyzed using the IFN-Secretion Assay (PE, Miltenyi Biotec) following manufacturers instruction.

Bibby J., Agarwal D., Freiwald T., Kunz N., Merle N.S., West E.E., Larochelle A., Chinian F., Mukherjee S., Afzali B., Kemper C., & Zhang N.R. (2022). Systematic Single Cell Pathway Analysis (SCPA) reveals novel pathways engaged during early T cell activation.

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