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Cd45ra percp cy5

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The CD45RA - PerCP-Cy5.5 is a fluorescent-conjugated antibody product used for flow cytometry applications. It targets the CD45RA cell surface antigen, which is expressed on a subset of T cells. The PerCP-Cy5.5 fluorochrome is used for signal detection and identification of the labeled cells.

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Lab products found in correlation

Cd4 apc efluor 780, by Thermo Fisher Scientific (3 mentions) Cd25 pe, by Thermo Fisher Scientific (2 mentions) Cd3 bv605, by BioLegend (2 mentions) Ccr7 pe, by BioLegend (2 mentions) Igd fitc, by BD (2 mentions) Pd 1 apc, by Thermo Fisher Scientific (2 mentions) Cd8 bv650, by Thermo Fisher Scientific (2 mentions) Cd19 bv450, by BD (2 mentions) Cd62l apc ef780, by Thermo Fisher Scientific (2 mentions) Cxcr5 af488, by BioLegend (2 mentions) Hla dr efluor450, by Thermo Fisher Scientific (2 mentions) Cxcr3 fitc, by BioLegend (2 mentions) Cd27 pe cy7, by Thermo Fisher Scientific (2 mentions) Cd127 apc, by Thermo Fisher Scientific (2 mentions) Histopaque 1077, by Merck Group (2 mentions) Lsrfortessa, by BD (2 mentions) Facsdiva software, by BD (2 mentions) Fcs express, by De Novo Software (1 mentions) Pe conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions) Cd69 fitc, by BD (1 mentions)

6 protocols using cd45ra percp cy5

1

Multiparametric Flow Cytometry Immunophenotyping

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Peripheral blood mononuclear cells were prepared for analysis by density centrifugation using Histopaque-1077 (Sigma-Aldrich). The following antibodies were used for flow cytometry immunophenotyping: CD3 – BV605 (Biolegend, San Diego, CA, USA), CD4 – APC-eFluor780 (eBioscience, San Diego, CA,USA), CD8 – BV650 (eBioscience, San Diego, CA,USA), CD25 – PE (eBioscience, San Diego, CA,USA), CD127 – APC (eBioscience, San Diego, CA,USA), CD45RA – PerCP-Cy5.5(eBioscience, San Diego, CA,USA, CD19 – BV450 (BD Bioscience, Franklin Lakes, NJ, USA), CD27 – PE-Cy7 (eBioscience, San Diego, CA,USA, CD62L – APC-eF780 (eBioscience, San Diego, CA,USA, CXCR3 – FITC (Biolegend, San Diego, CA, USA), CXCR5 – AF488 (Biolegend, San Diego, CA, USA), CCR7 – PE (Biolegend, San Diego, CA, USA), PD-1 – APC (eBioscience, San Diego, CA,USA), HLA-DR- eFluor450 (eBioscience, San Diego, CA, USA), IgD – FITC (BD Bioscience, Franklin Lakes, NJ, USA). Flow cytometry analysis was performed on a BD LSRFortessa (BD Bioscience) with FACS Diva software (BD Bioscience) for acquisition, then analysis was performed with FlowJo software (LLC).
Thaventhiran J.E., Allen H.L., Burren O.S., Rae W., Greene D., Staples E., Zhang Z., Farmery J.H., Simeoni I., Rivers E., Maimaris J., Penkett C.J., Stephens J., Deevi S.V., Sanchis-Juan A., Gleadall N.S., Thomas M.J., Sargur R.B., Gordins P., Baxendale H.E., Brown M., Tuijnenburg P., Worth A., Hanson S., Linger R., Buckland M.S., Rayner-Matthews P.J., Gilmour K.C., Samarghitean C., Seneviratne S.L., Sansom D.M., Lynch A.G., Megy K., Ellinghaus E., Ellinghaus D., Jorgensen S.F., Karlsen T.H., Stirrups K.E., Cutler A.J., Kumararatne D.S., Chandra A., Edgar J.D., Herwadkar A., Cooper N., Grigoriadou S., Huissoon A., Goddard S., Jolles S., Schuetz C., Boschann F., Lyons P.A., Hurles M.E., Savic S., Burns S.O., Kuijpers T.W., Turro E., Ouwehand W.H., Thrasher A.J, & Smith K.G. (2020). Whole genome sequencing of a sporadic primary immunodeficiency cohort. Nature, 583(7814), 90-95.
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2

Immunophenotyping of Thymocyte Subsets

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Surface immunophenotyping of thymocytes with unconjugated and directly conjugated antibodies was performed as previously described [15 (link),48 (link)]. Monoclonal antibodies (mAb) were procured from R&D (Minneapolis, MN, USA) (unconjugated anti-human S1PR1 (clone 218713, product #MAB2016), unconjugated IgG2b control), eBioscience (PE-conjugated anti-mouse IgG), eBioscience (CD45RA PerCP-Cy5.5, CD8 APC-eFluor 780, CD25 eFluor 450, CD62L eFluor 605NC, CD3 eFluor 650NC), and Becton Dickinson (CD69 FITC, CD27 APC, and CD4 PE-Cy7). For S1PR1 detection, cells were first stained with an unconjugated anti-human S1PR1 (or IgG2b isotype control), followed by a phycoerythrin-conjugated anti-mouse IgG antibody. Flow cytometry data were acquired on an LSRII or LSRII-HT analyzer (Becton Dickinson) and analyzed with FCS Express (Version 6, De Novo software). Gates were set based on isotype controls, unstained thymocytes, or fluorescence minus one (FMO) controls, as appropriate.
Resop R.S., Salvatore B., Kim S.J., Gordon B.R., Blom B., Vatakis D.N, & Uittenbogaart C.H. (2023). HIV-1 Infection Results in Sphingosine-1-Phosphate Receptor 1 Dysregulation in the Human Thymus. International Journal of Molecular Sciences, 24(18), 13865.
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3

Multiparametric Flow Cytometry Immunophenotyping

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Peripheral blood mononuclear cells were prepared for analysis by density centrifugation using Histopaque-1077 (Sigma-Aldrich). The following antibodies were used for flow cytometry immunophenotyping: CD3 – BV605 (Biolegend, San Diego, CA, USA), CD4 – APC-eFluor780 (eBioscience, San Diego, CA,USA), CD8 – BV650 (eBioscience, San Diego, CA,USA), CD25 – PE (eBioscience, San Diego, CA,USA), CD127 – APC (eBioscience, San Diego, CA,USA), CD45RA – PerCP-Cy5.5(eBioscience, San Diego, CA,USA, CD19 – BV450 (BD Bioscience, Franklin Lakes, NJ, USA), CD27 – PE-Cy7 (eBioscience, San Diego, CA,USA, CD62L – APC-eF780 (eBioscience, San Diego, CA,USA, CXCR3 – FITC (Biolegend, San Diego, CA, USA), CXCR5 – AF488 (Biolegend, San Diego, CA, USA), CCR7 – PE (Biolegend, San Diego, CA, USA), PD-1 – APC (eBioscience, San Diego, CA,USA), HLA-DR- eFluor450 (eBioscience, San Diego, CA, USA), IgD – FITC (BD Bioscience, Franklin Lakes, NJ, USA). Flow cytometry analysis was performed on a BD LSRFortessa (BD Bioscience) with FACS Diva software (BD Bioscience) for acquisition, then analysis was performed with FlowJo software (LLC).
Thaventhiran J.E., Allen H.L., Burren O.S., Rae W., Greene D., Staples E., Zhang Z., Farmery J.H., Simeoni I., Rivers E., Maimaris J., Penkett C.J., Stephens J., Deevi S.V., Sanchis-Juan A., Gleadall N.S., Thomas M.J., Sargur R.B., Gordins P., Baxendale H.E., Brown M., Tuijnenburg P., Worth A., Hanson S., Linger R., Buckland M.S., Rayner-Matthews P.J., Gilmour K.C., Samarghitean C., Seneviratne S.L., Sansom D.M., Lynch A.G., Megy K., Ellinghaus E., Ellinghaus D., Jorgensen S.F., Karlsen T.H., Stirrups K.E., Cutler A.J., Kumararatne D.S., Chandra A., Edgar J.D., Herwadkar A., Cooper N., Grigoriadou S., Huissoon A., Goddard S., Jolles S., Schuetz C., Boschann F., Lyons P.A., Hurles M.E., Savic S., Burns S.O., Kuijpers T.W., Turro E., Ouwehand W.H., Thrasher A.J, & Smith K.G. (2020). Whole genome sequencing of a sporadic primary immunodeficiency cohort. Nature, 583(7814), 90-95.
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4

Brachyury-specific T-cell Characterization

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The brachyury agonist-specific T-cell lines were examined for antigen-specificity and perforin production. T cells (2×105) were stained with FITC-conjugated anti-CD8 (BD) and either a PE-conjugated agonist-specific tetramer (NIH Tetramer Core Facility, Atlanta, GA) or a negative control tetramer (Beckman Coulter, Fullerton, CA). Cells stained for perforin production were surface stained with FITC-conjugated anti-CD8 (BD), then permeabilized using a Fixation/Permeabilization kit (eBioscience, San Diego, CA) according to the manufacturer’s instructions. Cells then underwent intracellular staining with PE-conjugated anti-perforin antibody or isotype control (BD). In addition, T-cell lines were stained with CD8-V421 (BD), CD45RA-PerCP-Cy5.5 (eBioscience), CCR7-AF700 (R&D Systems, Minneapolis, MN), CD27-PE (BD), Perforin-APC (BioLegend, San Diego, CA) and Ki67-FITC (BD), and 1×105 cells were captured on an LSRII (BD) and analyzed using FlowJo 9.0.1 software (Tree Star Inc, Ashland, OR).
Tucker J.A., Jochems C., Boyerinas B., Fallon J., Greiner J.W., Palena C., Rodell T.C., Schlom J, & Tsang K.Y. (2014). Identification and characterization of a cytotoxic T-lymphocyte agonist epitope of brachyury, a transcription factor involved in epithelial to mesenchymal transition and metastasis. Cancer immunology, immunotherapy : CII, 63(12), 1307-1317.
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5

Isolation of Naive T-Cell Subpopulations

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Human peripheral blood mononuclear cells were isolated from fresh whole blood by density gradient centrifugation using Histopaque (ρ = 1.077 g/cm 3 , Sigma, USA). The subpopulations of nTh (CD4 + CD45RO -) and nCTL (CD8 + CD45RO -) were isolated by negative magnetic immunoseparation using commercial kits series EasySep (Stemcell Technologies, UK). The purity of isolated cells was monitored by flow cytometry using a panel of fluorescent-labeled mAb: CD3-PE, CD45RO-PE-Cy7, CD45RA-PerCP-Cy5.5, and CD8-APC-eFluor780 (or CD4-APC-eFluor780) (eBioscience, USA). We verified that the subpopulations of nTh and nCTL T-cells were purified to more than 98% (Figure 1).
Filatova E.N., Anisenkova E.V., Presnyakova N.B, & Utkin O.V. (2016). DR3 regulation of apoptosis of naive T-lymphocytes in children with acute infectious mononucleosis. Acta microbiologica et immunologica Hungarica, 63(3).
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6

CD4+ T Cell Immunophenotyping and Activation

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For surface staining, cells were incubated with CD25 BV605 (clone 2A3; BD), CD4 Alexa Fluor 700 (clone RPA--T4; BD), CD45RA PerCP-Cy5.5 (clone HI100; eBioscience) at 4°C for 30 minutes. For analysis of total CTLA--4 and Foxp3 expression, cells were fixed and permeabilized with Foxp3 staining buffer (eBioscience) and incubated with Foxp3 allophycocyanin (clone 236A--E7; eBioscience) and CTLA--4 phycoerythrin (clone BNI3; BD). Cells were acquired on a BD LSRII cytometer and the data analyzed using FlowJo software (TreeStar). In some experiments relative expression of CTLA--4 or CD25 was calculated based on: level of expression (MFI) in memory Treg (CD45RA--Foxp3+)/ level of expression (MFI) in naïve conventional CD4 cells (CD4+CD45RA+ Foxp3--) 2.5 T--cell stimulation CD4 T cells were resuspended at 1 × 10 6 /mL in RPMI 1640 with 10% fetal bovine serum, 2 mM l--glutamine, 1% penicillin, and 1% streptomycin. 96,000 Tells were stimulated with 0.5µg/ml anti--CD3 plus 72,000 CHO--cells expressing the CD80 ligand as previously described [24] . Cells were cultured in a 96--well round--bottomed plate at 37°C, 95% humidity, and 5% CO2.
Hou T.Z., Olbrich P., Soto J.M.L., Sanchez B., Moreno P.S., Borte S., Stauss H.J., Burns S.O., Walker L.S.K., Pan-Hammarström Q., Hammarström L., Sansom D.M, & Neth O. (2018). Study of an extended family with CTLA-4 deficiency suggests a CD28/CTLA-4 independent mechanism responsible for differences in disease manifestations and severity. Clinical immunology (Orlando, Fla.), 188.
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