Cd45ra ecd

Manufactured by Beckman Coulter

Sourced in United States, France

The CD45RA-ECD is a monoclonal antibody that binds to the CD45RA isoform of the CD45 protein. CD45 is a transmembrane protein tyrosine phosphatase expressed on the surface of all nucleated hematopoietic cells. The CD45RA isoform is specifically expressed on naive T cells and a subset of memory T cells. The ECD (Phycoerythrin-Texas Red®-X) fluorescent label allows for the detection and analysis of CD45RA-positive cells using flow cytometry.

Automatically generated - may contain errors

Lab products found in correlation

Live dead aqua, by Thermo Fisher Scientific (2 mentions) Trucount tube, by BD (2 mentions) Aqua fluorescent reactive dye, by Thermo Fisher Scientific (1 mentions) Facs lsr 2, by BD (1 mentions) Cd4 alexafluor700, by BD (1 mentions) Navios flow cytometer, by Beckman Coulter (1 mentions) Cd3 pe cy7, by BD (1 mentions) Cd25 pe, by BD (1 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions) Foxp3 alexafluor488, by BD (1 mentions) Cd62l percp cy5, by BioLegend (1 mentions) Cd14 pacific orange, by Thermo Fisher Scientific (1 mentions) Cd3 pacific blue, by BD (1 mentions) Ccr7 bv650, by BD (1 mentions) Cd95 fitc, by BD (1 mentions) Cd57 pb, by BioLegend (1 mentions) Cd49d pecy7, by BioLegend (1 mentions) Aria 3, by BD (1 mentions) Allprep dna rna micro kit, by Qiagen (1 mentions) Cd4 apc cy7, by BD (1 mentions)

Spelling variants of this product

Anti-CD45RA-ECD (4 citations) CD45RA ECD (3P, (2 citations) Anti-CD45RA–ECD (clone 2H4LDH11LDB9; (2 citations) ECD-conjugated anti-CD45RA (clone 2H4) (2 citations)

10 protocols using cd45ra ecd

Immunophenotyping of Regulatory T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Whole blood, PBMC, and nTreg staining was performed using the FoxP3 Staining Buffer Set purchased from eBioscience (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's recommendations. The following antibodies were used for wholeblood staining: CD14-PacificOrange (Invitrogen, Darmstadt, Germany); CD3-PacificBlue, CD4-AlexaFluor700, CD25-PE (BD Biosciences, Heidelberg, Germany); CD127-APC-AlexaFluor780, CD62L-PerCP-Cy5.5 (BioLegend, San Diego, CA); CD45RA-ECD (Beckman Coulter, Krefeld, Germany); and FoxP3-AlexaFluor488 (BD Pharmingen, Heidelberg, Germany). The following antibodies were used for PBMC stainings: CD3-PE-Cy7, CD4-AlexaFluor700, CD25-PE (BD Biosciences), CD14-PacificOrange (Invitrogen), CD127-APC-AlexaFluor780, CD45RA-ECD (Beckman Coulter); FoxP3-AlexaFluor488, CD137-PE-Cy5 (BD Biosciences), and CD154-PacificBlue (BioLegend). Dead cells were routinely detected by staining with aqua fluorescent reactive dye (Invitrogen). Data acquisition was performed on a FACS LSRII (BD Biosciences) or Navios flow cytometer (Beckman Coulter). FlowJo software V8 (Treestar) was used for further analysis. c l i n i c a l i n v e s t i g a t i o n S Landwehr-Kenzel et al.: nTregs in kidney transplantation nTregs were defined as CD4 þ CD25 high FoxP3 þ cells. By Boolean gating, non-Treg cells were identified and considered Tconv.

Landwehr-Kenzel S., Zobel A., Hoffmann H., Landwehr N., Schmueck-Henneresse M., Schachtner T., Roemhild A, & Reinke P. (2018). Ex vivo expanded natural regulatory T cells from patients with end-stage renal disease or kidney transplantation are useful for autologous cell therapy. Kidney international, 93(6).

+ Open protocol

+ Expand

Isolation and Characterization of Naive T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

After thawing, PBMCs were stained with CCR7 BV650 (Clone 3D12; BD Biosciences), CD95 FITC (Clone DX2; BD Biosciences), CD27 PE (Clone M-T271; BD Biosciences), CD8 APC (Clone RPA-T8; BD Biosciences), CD4 APC-Cy7 (Clone RPA-T4; BD Biosciences), CD45RA ECD (Clone IM271111; Beckman Coulter), CD57 PB (Clone HCD57; Biolegend), CD49d PeCY7 (Clone 9F10; Biolegend), and eFluor 506 for viability (Biolegend). Naive CD4⁺ and CD8⁺ T cells, defined as CD45RA⁺, CD27⁺ CCR7⁺, CD95⁻, CD49d⁻, were sorted on an ARIAIII (Becton Dickinson). DNA and RNA from sorted cells were extracted using AllPrep DNA/RNA Micro kit (Qiagen) according to manufacturer instruction.

Nicoli F., Clave E., Wanke K., von Braun A., Bondet V., Alanio C., Douay C., Baque M., Lependu C., Marconi P., Stiasny K., Heinz F.X., Muetsch M., Duffy D., Boddaert J., Sauce D., Toubert A., Karrer U, & Appay V. (2022). Primary immune responses are negatively impacted by persistent herpesvirus infections in older people: results from an observational study on healthy subjects and a vaccination trial on subjects aged more than 70 years old. EBioMedicine, 76, 103852.

+ Open protocol

+ Expand

Multiparameter flow cytometric analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

For the analysis of OX40L expression, whole blood samples were stained with anti-CD14-PC5, CD16-FITC, CD11c-APC, HLA-DR-PC7, and OX40L-PE mAbs, and red blood cells were lysed with Versalyse (Beckman Coulter). For the analysis of blood Tfh cells, whole blood samples were stained with anti-CXCR5-AF488, CCR6-PE, CXCR3-PC5, CCR4-PC7, CD3-AF700, CD8-APCH7, CD4-Pacific Blue (all from Becton Dickinson), CD45RA-ECD (Beckman Coulter), ICOS-APC (Biolegend) and CD45-Pacific Orange (Invitrogen). Data were collected using a BD LSR II instrument (BD Biosciences) and analyzed with Flowjo software (Tree Star Inc.).

Jacquemin C., Schmitt N., Contin-Bordes C., Liu Y., Narayanan P., Seneschal J., Maurouard T., Dougall D., Davizon E.S., Dumortier H., Douchet I., Raffray L., Richez C., Lazaro E., Duffau P., Truchetet M.E., Khoryati L., Mercié P., Couzi L., Merville P., Schaeverbeke T., Viallard J.F., Pellegrin J.L., Moreau J.F., Muller S., Zurawski S., Coffman R.L., Pascual V., Ueno H, & Blanco P. (2015). OX40 Ligand Contributes to Human Lupus Pathogenesis by Promoting T follicular Helper Response. Immunity, 42(6), 1159-1170.

+ Open protocol

+ Expand

Detailed Immunophenotyping of T-cell Subsets

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following antibodies were used for T-cell phenotyping: CD8-BV650 (BD), CD4-BV605 (BD Biosciences), CD3-Alexa Fluor700 (BD Biosciences), CD95-BV711 (BD Biosciences), CD45RO-PerCPCy5.5 (BD Biosciences), CD25-APC-Cy7 (BD Biosciences), CD127-BV421 (BioLegend), CCR7-PE-Cy7 (BioLegend), CD45RA-ECD (Beckman Coulter), PD-1-BV785 (BioLegend), LAG3-FITC (R&D Systems), and TIM3-APC (R&D Systems). The dead cell exclusion stain (Live/Dead Aqua) was purchased from Invitrogen. To detect transduced NY-ESO-1^c259TCR-expressing cells, PE-conjugated pentamer reagents specific for the HLA-A*02:01 SLLMWITQC complex (ProImmune) were used at the manufacturer’s recommended concentrations.

D'Angelo S.P., Melchiori L., Merchant M.S., Bernstein D., Glod J., Kaplan R., Grupp S., Tap W.D., Chagin K., Binder G.K., Basu S., Lowther D.E., Wang R., Bath N., Tipping A., Betts G., Ramachandran I., Navenot J.M., Zhang H., Wells D.K., Winkle E.V., Kari G., Trivedi T., Holdich T., Pandite L., Amado R, & Mackall C.L. (2018). Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1c259T Cells in Synovial Sarcoma. Cancer discovery, 8(8), 944-957.

+ Open protocol

+ Expand

Multiparameter Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following monoclonal antibodies (mAbs) were used in different combinations. CD8-PB, CD3-APC-H7, PD-1-PECy7, PD-1-PB, IFN-γ-AF700, IL-2-PE, CD4-PB, granzyme B-AF700, perforin-APC were purchased from Becton Dickinson (BD, San Diego, CA), CD45RA-ECD, CD4-ECD from Beckman Coulter (Fullerton, CA, USA), CCR7-FITC from R&D Systems (Minneapolis, MN, USA), 2B4-PECY5.5, CD160-APC, CD160-PE from BioLegend (San Diego, CA, USA), CD4-eFluor650NC, CD8-eFluor625NC from eBioscience.

Viganò S., Banga R., Bellanger F., Pellaton C., Farina A., Comte D., Harari A, & Perreau M. (2014). CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression. PLoS Pathogens, 10(9), e1004380.

+ Open protocol

+ Expand

Quantifying CMV-Specific CD8+ T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

The Dextramer CMV kit (Immudex, Copenhagen, Denmark) was used according to the manufacturer's instructions to quantify the CMV-specific CD8+ T cells. The CMV-MHC class I dextramers used, which covered most HLA specificities, were A:0101, A:0201, A:0301, A:2402, B:0702, B:0801, and B:3501. Samples were acquired using a Gallios flow cytometer (Beckman Coulter, Pasadena, CA, USA) and analyzed using Kaluza Analysis software (Beckman Coulter, Pasadena, CA, USA). A whole-blood control for lymphocyte subset enumeration was used in all sessions (BD^TM Multi-Check control, BD Biosciences, San Jose, CA, USA). Absolute CD8+ T cells number and percentage were calculated using Trucount Tubes (BD Biosciences, San Jose, CA, USA).
For CD8+ T cell phenotyping, whole-EDTA anticoagulate was obtained by venipuncture. Peripheral blood mononuclear cells were obtained by Ficoll–Paque density-gradient centrifugation and directly analyzed. Naïve (CCR7+CD45RA+), central memory (CM) (CCR7+CD45RA–), effector memory (EM) (CCR7–CD45RA−), and effector memory RA (EMRA) (CCR7–CD45RA+) CD8+ T cells were assessed by flow cytometry using the following antibodies: CD3 (PerCP; BD Biosciences, San Jose, CA, USA), CD8 (FITC; BD Biosciences, San Jose, CA, USA), CD45RA (ECD; Beckman Coulter, Pasadena, CA, USA), and CCR7 (APC; BioLegend, San Diego, CA, USA).

Vidal-Castiñeira J.R., Corte-Iglesias V., Sobrino-Diaz L., Pérez-Fernández S., Melón S., López-Larrea C, & Díaz-Corte C. (2019). Effect of Type of Dialysis on CMV-Specific CD8+ T Cells in Kidney Transplant Candidates. Frontiers in Immunology, 10, 1680.

+ Open protocol

+ Expand

T cell Profiling and Exhaustion Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

T cell sub-population profiling studies on days 14 and 21 were analyzed using CD3-PC7 (Beckman Coulter, 6607100), CD4-APC-A700 (Beckman Coulter, B10824), CD8-PC5.5 (Beckman Coulter, B21205), EGFR-A488 (R&D Systems, FAB10951G), CD45RA-ECD (Beckman Coulter, IM2711U), CD45RO-PE (Miltenyi Biotech,130-113-559), CD27-APC-A750 (Beckman Coulter, B12701), and CD62L-APC (Miltenyi Biotech, 130-113-617) antibodies by Cytoflex Flow Cytometer analysis. Exhaustion profiling studies on days 14 and 21 were analyzed using CD3-PC7 (Beckman Coulter, 6607100), CD4-APC-A700 (Beckman Coulter, B10824), CD8-PC5.5 (Beckman Coulter, B21205), EGFR-A488 (R&D Systems, FAB10951G), CD279 (PD1)-PE (Miltenyi Biotech, 130-117-384), CD366 (TIM3)—APC (Invitrogen, Waltham, MA, USA, 17-3109-42), and CD223 (LAG-3)—APC-eFluor 780 (Invitrogen, 47-2239-42) antibodies by Cytoflex Flow Cytometer analysis.

Gulden G., Sert B., Teymur T., Ay Y., Tiryaki N.N., Mishra A.K., Ovali E., Tarhan N, & Tastan C. (2023). CAR-T Cells with Phytohemagglutinin (PHA) Provide Anti-Cancer Capacity with Better Proliferation, Rejuvenated Effector Memory, and Reduced Exhausted T Cell Frequencies. Vaccines, 11(2), 313.

+ Open protocol

+ Expand

Comprehensive T-cell Immunophenotyping by Flow Cytometry

Check if the same lab product or an alternative is used in the 5 most similar protocols

CD4 and CD8 T-lymphocyte phenotypes were determined on fresh whole blood, using combinations of the following antibodies: CD28-FITC, CD4-RD1, CD45RA-ECD, CD45RO-ECD, CD62L-PC5, CD27-PC5, CD4-PC7, and CD8-PC7 (Beckman Coulter, Villepinte, France), as described in [18 (link)]. CD4_N levels were defined as the percentage of CD4 T lymphocytes that were CD45RA⁺CD62L⁺, CD8_N levels as the percentage of CD8 T lymphocytes that were CD45RO^-CD28⁺CD27⁺. For 57 patients, frozen PBMCs were available for the quantification of CD4_RTE, with Live-Dead-Aqua (Life Technologies, Saint-Aubin, France) labeling of dead cells, and the following antibodies: CD3-ECD and CD31-FITC (Beckman Coulter), CD4-APC-efluor780 (e-bioscience, Paris, France), CD45RA-V450 and CD197-PC7 (BD Biosciences, Rungis, France). CD4_RTE levels were defined as the percentage of naive CD45RA⁺CCR7⁺CD4⁺ T lymphocytes positive for CD31.

Le Chenadec J., Scott-Algara D., Blanche S., Didier C., Montange T., Viard J.P., Dollfus C., Avettand-Fenoel V., Rouzioux C., Warszawski J, & Buseyne F. (2015). Gag-Specific CD4 and CD8 T-Cell Proliferation in Adolescents and Young Adults with Perinatally Acquired HIV-1 Infection Is Associated with Ethnicity — The ANRS-EP38-IMMIP Study. PLoS ONE, 10(12), e0144706.

+ Open protocol

+ Expand

Comprehensive Immune Cell Phenotyping

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMC (1–4×10⁶ cells/tube) were stained with monoclonal antibodies to B cell markers (CD19-AF700, IgM-PerCP-Cy5.5, IgD-PE-Cy7 (Biolegend), CD38-FITC, CD21-PE, CD10-PE-CF594, CD40-APC, CD86-BV421 (BD Pharmingen)) and T cell markers (CD3-AF700, CD4PacBlue (Biolegend), CD8-APC-AF750 (Invitrogen), CD45RA-ECD, CD27-PE-Cy5 (Beckman Coulter), CD27-BV650, CD38-FITC, HLA-DR-PE-Cy7, PD-1-APC, CXCR5-PE (R&D Systems)) for 40 minutes at room temperature. Stained cells were subsequently washed twice and fixed in 1% paraformaldehyde for 10 minutes at 4°C. Data was acquired within 4 hours using a BD LSRII flow cytometer (BD Biosciences – Immunocytometry Systems, San Jose, CA). Data were analyzed using Flow Jo Software (Tree Star Inc., Ashland, OR) with the gating scheme as per Supplemental Figure 1.

Nicholson L.K., Pratap H., Bowers E., Gunzburger E., Bandi S., Gardner E.M., Palmer B.E., Wright T., Kittelson J, & Janoff E.N. (2018). Effective B cell Activation in vitro during Viremic HIV-1 Infection with Surrogate T cell Stimulation. Immunobiology, 223(12), 839-849.

+ Open protocol

+ Expand

Immunophenotyping of T and B Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

After isolation, PBMCs were stained with the following fluorochrome-conjugated antibodies: CXCR3 (CD183) FITC, CCR6 PE-Vio770 (Miltenyi Biotec, Paris, France); CXCR5 (CD185) PE (eBioscience, ThermoFisher, Villebon, France); CD45RA ECD, PD-1 (CD279) PECY5.5, ICOS (CD278) APC, CD3 AA750 (Beckman Coulter, Villepinte, France); CD45 BV510, CD4 BV650, HLA-DR BV786 (BD Biosciences, Rungis, France). For B-lymphocyte analysis, the following fluorochrome-conjugated antibodies were used: IgD FITC, CD21 BV450, CD45 BV510, IL-21R APC (BD Biosciences); CD27 PE, CD24 ECD, CD38 PECY5.5, CD19 PC7 (Beckman Coulter); CD5 AA700 (Biolegend, Ozyme, Montigny-le-Bretonneux). T-lymphocyte and B-lymphocyte absolute numbers were determined using 50 µL of whole blood from patients and HC using Trucount tubes (BD Biosciences). Samples were stained with the following antibodies: CD19 FITC, CD45 ECD, CD3 AA750 (Beckman Coulter) for 15 min and then incubated for 15 min with 450 µL BD FACS Lysis Solution and subsequently analysed by flow cytometry. Compensation beads were used for compensation setting (VersaComp; Beckman Coulter). Cells were analysed on a Cytoflex flow cytometer (Beckman Coulter). Data were analysed using Kaluza V.5.1 software (Beckman Coulter).

Ricard L., Jachiet V., Malard F., Ye Y., Stocker N., Rivière S., Senet P., Monfort J.B., Fain O., Mohty M., Gaugler B, & Mekinian A. (2019). Circulating follicular helper T cells are increased in systemic sclerosis and promote plasmablast differentiation through the IL-21 pathway which can be inhibited by ruxolitinib. Annals of the rheumatic diseases, 78(4).

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!