Cd45ra apc

Manufactured by Thermo Fisher Scientific

Sourced in United States

The CD45RA APC is a fluorescently labeled monoclonal antibody that binds to the CD45RA cell surface antigen. It is used to identify and study T cells, B cells, and other immune cells in flow cytometry applications.

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Close spelling variants of this product

CD45RA-APC-eFluor780 (HI100) (3 citations) CD45RA APC-eFluor 780 (clone HI100; (2 citations) CD45RA APC-Alexa eFluor 780 (2 citations) Anti-mouse CD45RA-APC/ eFluor780 (2 citations)

8 protocols using cd45ra apc

CAR T Cell Immunophenotyping and Transduction

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Immunophenotyping of the apheresis was conducted with the following fluorophore-conjugated antibodies: CD3-phycoerythrin (PE) (Beckman Coulter), CD8-PE-Cy7 (Invitrogen), CD14-allophycocyanin (APC) (eBioscience), and CD45-fluorescein isothiocyanate (FITC) (Beckman Coulter). In-process transduction efficiency of the CD19-targeted CAR T cells was evaluated with the CD3-APC (Invitrogen) and biotinylated goat-anti-mouse Fab (Jackson Immunoresearch Lab) followed by PE-conjugated streptavidin (MP Biomedicals). In-process transduction efficiency of the BCMA-targeted CAR T cells was evaluated with the CD3-APC (Invitrogen) and BCMA-Fc-APC.¹² (link) The effector memory and central memory immunophenotyping was conducted using the following monoclonal antibodies: CD27-APC, CD28-FITC, CD62L-FITC, CCR7-FITC, CD45RA-APC (Invitrogen), and CD127-eFlour450 (eBioscience). Dead cells were excluded from analysis using either 7AAD or DAPI staining. Flow data acquisition was performed on an LSRII (BD Biosciences), and data analysis was performed using FlowJo Software (Tree Star).

Wang X., Borquez-Ojeda O., Stefanski J., Du F., Qu J., Chaudhari J., Thummar K., Zhu M., Shen L.B., Hall M., Gautam P., Wang Y., Sénéchal B., Sikder D., Adusumilli P.S., Brentjens R.J., Curran K., Geyer M.B., Mailankhody S., O’Cearbhaill R., Park J.H., Sauter C., Slovin S., Smith E.L, & Rivière I. (2021). Depletion of high-content CD14+ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing. Molecular Therapy. Methods & Clinical Development, 22, 377-387.

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Multiparameter Immunophenotyping of Immune Cells

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Whole blood was stained with 3 panels of antibodies incubated for 20 min followed by fixation and RBC lysis using FACS lysis buffer (BD) in accordance with prescribed. Panel I consisted of integrin α4-PE (Invitrogen), integrin β7-FITC (Biolegend) CD19-PECy7 (BD Pharmingen), CD14APC (eBiosciences). Panel II consisted of integrin α4-PE (eBiosciences), integrin β7-FITC (Biolegend), CD3-PECy7 (BD Pharmingen), CD56APC (BD Pharmingen). Cells stained with both these four color panels were acquired on BD accuri C6 and analyzed using BD accuri C6 software (BD Biosciences). Panel III consisted of 5 antibodies viz. integrin β7-FITC (Biolegend), CCR7-PE (eBiosciences), CD45RA APC (Invitrogen), CD3-PECy7, CD4-PECF594 (BD Horizon). Cells stained with Panel III were acquired on FACS Aria fusion and analyzed using FlowJo VX (TreeStar, Ashland, OR,USA). Cells from the two cytobrushes were pooled and filtered through a 100-mm filter followed by staining with integrin α4-PE, integrin β7-FITC (Biolegend) and CD3-APC (BD). Viability dye 7AAD (eBiosciences) was added before acquiring the cells on BD accuri C6 flow cytometer.

Kasarpalkar N.J., Bhowmick S., Patel V., Savardekar L., Agrawal S., Shastri J, & Bhor V.M. (2021). Frequency of Effector Memory Cells Expressing Integrin α4β7 Is Associated With TGF-β1 Levels in Therapy Naïve HIV Infected Women With Low CD4+ T Cell Count. Frontiers in Immunology, 12, 651122.

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Multiparametric Flow Cytometry Analysis of PBMCs

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PBMCs were thawed, stained with 5 μl of the HLA-A*02:01 dextramer conjugated to PE for 10 minutes at room temperature, followed by the addition of 100 μl of a mastermix of anti-membrane marker mAbs containing LIVE/DEAD fixable aqua stain (Molecular Probes, Invitrogen), CD3 ECD (Beckman Coulter), CD4 BV605 and CCR7 Pacific blue (Biolegend), CD8 Alexa Fluor 700, CD14 PE-Cy7, CD16 PE-Cy7, CD19 PE-Cy7, CD45RA APC, CD57 FITC, TIGIT PerCP-eFluor710, PD-1 APC-eFluor780 (eBiosciences) and CD27 Qdot 655 (Life Technologies). The cells were incubated at 4°C for a further 20 minutes, washed and fixed with 1% paraformaldehyde in PBS prior to running on an LSRII flow cytometer (Becton-Dickinson).

Moyo N., Borthwick N.J., Wee E.G., Capucci S., Crook A., Dorrell L, & Hanke T. (2017). Long-term follow up of human T-cell responses to conserved HIV-1 regions elicited by DNA/simian adenovirus/MVA vaccine regimens. PLoS ONE, 12(7), e0181382.

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Comprehensive Immune Phenotyping of PBMCs

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PBMCs were thawed, incubated with 5 μl of the HLA-A*02:01/YQYMDDLV tetramer (NIH Tetramer Core Facility) conjugated to PE for 10 min at room temperature, followed by the addition of 100 μl of a mastermix of anti-membrane marker mAbs containing LIVE/DEAD fixable aqua stain (Molecular Probes, Invitrogen), CD3 ECD (Beckman Coulter), CD4 BV605 and CCR7 Pacific blue (Biolegend), CD8 Alexa Fluor 700, CD14 PE-Cy7, CD16 PE-Cy7, CD19 PE-Cy7, CD45RA APC, CD57 FITC, TIGIT PerCP-eFluor710, PD-1 APC-eFluor780 (eBiosciences) and CD27 Qdot 655 (Life Technologies). The cells were incubated at 4°C for a further 20 min, washed and fixed with 1% paraformaldehyde in PBS prior to running on an LSRII flow cytometer (Becton-Dickinson).

Borthwick N.J., Lane T., Moyo N., Crook A., Shim J.M., Baines I., Wee E.G., Hawkins P.N., Gillmore J.D., Hanke T, & Pepys M.B. (2018). Randomized phase I trial HIV-CORE 003: Depletion of serum amyloid P component and immunogenicity of DNA vaccination against HIV-1. PLoS ONE, 13(5), e0197299.

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Isolation and Characterization of CD8+ T Cells

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Blood samples were collected at the Blood Centers of the Pacific in San Francisco, CA. All donors were kept anonymous and provided written informed consent. Total human CD8⁺ T cells were enriched via negative selection using the RosetteSep human CD8⁺ T cell enrichment cocktail (15063; Stemcell). The following antibodies were used to stain peripheral blood mononuclear cells or enriched CD8⁺ naive, CD8⁺CD28⁺ memory, and CD8⁺CD28⁻ T cells: CD3-PECy5 (555334; BD Biosciences) and CD3-APC-H7 (560176; BD Biosciences), CD8-V450 (560347; BD Biosciences), CD28-PE (12–0289; eBioscience), CD45RA-APC (17–0458; eBioscience), and CCR7-PECy7 (557648; BD Biosciences). Flow cytometry was performed on an LSRII or Calibur DxP8 flow cytometer (both BD Biosciences) and was analyzed with FlowJo software. Sorting was performed on an ARIA II (BD Biosciences).

Jeng M.Y., Hull P.A., Fei M., Kwon H.S., Tsou C.L., Kasler H., Ng C.P., Gordon D.E., Johnson J., Krogan N., Verdin E, & Ott M. (2018). Metabolic reprogramming of human CD8+ memory T cells through loss of SIRT1. The Journal of Experimental Medicine, 215(1), 51-62.

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Comprehensive Immune Cell Phenotyping

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Antibodies used for flow cytometry analysis included: CD2-Tri-Color (TC); CD4-Phycoerythrin (PE) and CD4Allophycocyanin (APC); CD28-Fluorescein Isothiocyanate (FITC); CD8-TC; CD19-APC; CD45RA-APC; CD16-FITC from Life Technologies (Grand Island, NY); CD14-PE; CD27-PE; and IgM-FITC from BD Biosciences (San Jose, CA). Freshly thawed PBMCs from each visit were stained with three to five antibodies: T cells (CD2, CD4, CD8, CD45RA, and CD28); B cells (CD19, IgM, and CD27); NK cells (CD16). The data were collected on a BD FACSCalibur or BD FACSCanto II, and analyzed by Cell-Quest (BD Biosciences) and FlowJo. The gating strategies were presented in Additional file 1: Figure S1.

Lin Y., Kim J., Metter E.J., Nguyen H., Truong T., Lustig A., Ferrucci L, & Weng N.P. (2016). Changes in blood lymphocyte numbers with age in vivo and their association with the levels of cytokines/cytokine receptors. Immunity & Ageing : I & A, 13, 24.

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Flow Cytometry Analysis of PBMCs

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Antibodies used for flow cytometry analysis included: CD2–Tri-Colour (TC); CD4–phycoerythrin (PE) and CD4–allophycocyanin (APC); CD28–FITC; CD8–TC; CD19–APC; CD45RA–APC from Life Technologies; CD14–PE; CD27–PE and IgM–FITC from BD Biosciences. Freshly thawed PBMCs of each visit were stained with 3–4 antibodies: T-cells (CD2, CD4, CD8, CD45RA and CD28); B-cells (CD19, IgM and CD27); monocytes (CD14). The data were collected on a BD FAC-SCalibur or BD FACSCanto II and analysed by Cell-Quest (BD Biosciences).

Lin Y., Damjanovic A., Metter E.J., Nguyen H., Truong T., Najarro K., Morris C., Longo D.L., Zhan M., Ferrucci L., Hodes R.J, & Weng N.P. (2015). Age-associated telomere attrition of lymphocytes in vivo is co-ordinated with changes in telomerase activity, composition of lymphocyte subsets and health conditions. Clinical science (London, England : 1979), 128(6), 367-377.

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Lentiviral Production and Immune Profiling

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HEK 293-FT (Life Technologies), a packaging cell line used to produce high-titer lentivirus supernatants, were cultured in complete Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum(FBS, Gibco, USA) containing 0.1 mM MEM Non-Essential Amino Acids, 1 mM sodium pyruvate and 2 mM Glutamax (Life Technologies, USA) at 37 °C with 5% CO2.
Flow cytometry staining antibodies were shown as below: CD3-BUV395(Clone: UCHT1), CD4-PE-CF594(Clone: RPA-T4), CD8-PE-Cy7(Clone: RPA-T8), PD-1-BUV737(Clone: EH12.1), CCR7-PE(Clone: 150503), CD45RA-APC(Clone: HI100), CD137-APC(Clone: 4B4–1), CD107A-AF647(Clone: H4A3), CD107b-AF647(Clone: H4B3), Fixable Viability Stain 780 (FVS780). All antibodies were from BD Biosciences, except for anti-mouse TCR-β constant region (clone H5–597, eBioscience, USA). The blocking antibody against HLA class I were from eBioscience (clone: W6/32).

Tan Q., Zhang C., Yang W., Liu Y., Heyilimu P., Feng D., Xing L., Ke Y, & Lu Z. (2019). Isolation of T cell receptor specifically reactive with autologous tumour cells from tumour-infiltrating lymphocytes and construction of T cell receptor engineered T cells for esophageal squamous cell carcinoma. Journal for Immunotherapy of Cancer, 7, 232.

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