Cd45ra apc

Manufactured by BioLegend

Sourced in United States

CD45RA-APC is a fluorochrome-conjugated antibody that binds to the CD45RA surface antigen, which is expressed on a subset of T cells and B cells. It can be used in flow cytometry applications to identify and quantify these cell populations.

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CD45RA-APC-Cy7 CD45RA HI100 APC-CY7 304128 CD45RA APC (clone HI100, Anti-CD45RA-APC-Cy7 Anti-CD45RA-APC-Cy7 (HI100), APC anti-human CD45RA Antibody APC anti-human CD45RA APC/Cy7 anti-human CD45RA Anti-CD45RA-APC

17 protocols using cd45ra apc

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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Phenotyping of CAR-T Cell Surface Markers

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Cell surface proteins on CAR-T cells were stained with the following antibodies: CD4-APC/H7, CD8a-PE/Cy7, CCR7-PerCP/Cy5.5, CXCR3-AF488, CD69-FITC, CD3-APC (all from BD Biosciences, San Jose, CA, USA); CD8a-FITC, CD28-FITC, CD27-PE, LAG-3-PE, TIM-3-FITC, TIGIT-PerCP/Cy5.5, CD45RA-APC (all from Biolegend, San Diego, CA, USA); PD-1-APC (eBioscience, Thermo Fisher Scientific, San Diego, CA, USA). To determine CAR expression, cells were labeled with a recombinant Fc-tagged BCMA protein (Enzo Life Sciences, Farmingdale, NY, USA) followed by a BV421-conjugated anti-Fc antibody (Biolegend). Samples prepared from mouse organs were treated with Fc block (BD Biosciences) prior to staining. Cells were washed and resuspended in 1% (v/v) FCS in PBS or 1% (v/v) paraformaldehyde prior to acquisition. Live cells were gated based on forward and side scatter.
For intracellular staining of γH2AX, surface-stained cells were fixed in 1% (v/v) paraformaldehyde, permeabilized in 90% (v/v) methanol at −20 °C overnight, and then stained with PE-conjugated anti-H2AX (pS139) antibody (BD Biosciences). Immediately prior to analysis, cells were washed and resuspended in 1% (v/v) FCS in PBS containing 3.3 µg/mL 7-AAD.
All flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software, version 7.6.2 (TreeStar, Ashland, OR, USA).

Battram A.M., Bachiller M., Lopez V., Fernández de Larrea C., Urbano-Ispizua A, & Martín-Antonio B. (2021). IL-15 Enhances the Persistence and Function of BCMA-Targeting CAR-T Cells Compared to IL-2 or IL-15/IL-7 by Limiting CAR-T Cell Dysfunction and Differentiation. Cancers, 13(14), 3534.

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Comprehensive T-cell Immunophenotyping Protocol

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Flow cytometry antibodies used for surface marker staining are listed as follows. Antibodies from BD Biosciences include: CD3‐BUV395 (Clone UCHT1; cat #563 546), CD4‐BV786 (Clone SK3; cat #563 877) and CD8‐PE‐Cy7 (Clone SK1; cat #335 787). Antibodies from Biolegend include: CD3‐FITC (Clone UCHT1; cat #300 406), CCR7‐PE (Clone G043H7; cat #353 204), CD45RA‐APC (Clone HI100; cat #304 112), CD45RO‐Pacific Blue (Clone UCHL1; cat #304 215),CD27‐Pacific Blue (Clone M‐T271; cat #356 413), CD28‐FITC (Clone CD28.2; cat #302 906), CD28‐PE (Clone CD28.2; cat #302 907), CD95‐FITC (Clone DX2; cat #305 605), CD127‐Brilliant Violet 785 (Clone A019D5; cat #351 329), TIM‐3‐Pacific Blue (Clone F38‐2E2; cat #345 041), CD57‐FITC (Clone HNK‐1; cat #359 603), LAG‐3‐Brilliant Violet 785 (Clone 11C3C65; cat #369 321) and human TruStain FcX reagent (Cat #422 302). Antibodies for intracellular staining include granzyme B‐PE (BD Biosciences, GB11; cat #561 142), IFN‐γ‐FITC (Miltenyi Biotec, cat #130‐090‐433), IL‐2‐PE (Miltenyi Biotec, cat #130‐090‐487) and TNF‐α‐APC (Miltenyi Biotec, cat #130‐091‐267).

Wu T., Tan J.H., Sin W., Luah Y.H., Tan S.Y., Goh M., Birnbaum M.E., Chen Q, & Cheow L.F. (2023). Cell Granularity Reflects Immune Cell Function and Enables Selection of Lymphocytes with Superior Attributes for Immunotherapy. Advanced Science, 10(28), 2302175.

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Profiling CMV- and IAV-specific CD8+ T cells

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The procedure of flow cytometric analysis of CMV- and IAV-specific CD8⁺ T cells prior to and post in vitro stimulation was described [40 (link)]. Briefly, freshly PMBCs were stained with fluorescently labeled dextramers or tetramers specific to CMV-pp65 (NLVPMVATV) and IAV-M1 (GILGFVFTL) (Immudex, Copenhagen, Denmark and NIAID tetramer core) first at room temperature for 20 min; followed by staining of cell surface markers including antibody against CD3, CD8, CD62L, CD45RA, CD95, CD27, CD28 CD70, CD127, and CD69 at 4°C for 30 min. Cells were washed again with FACS buffer (Hanks solution with 0.3% Sodium Azide), and then fixed immediately with 3% formaldehyde and 1% FBS in FACS buffer. Fixed cells were further stained with intracellular markers (perforin and granzyme B) at 4°C for 30 min. Stained cells were collected by BD_Symphony and were analyzed using FlowJo version 7.6.5 software.
Antibodies (CD3-BV570, CD62L-FITC, CD62L-PE-Cy7, CD45RA-PE-Cy7, CD45RA-APC, CD95-PE-Cy5, CD27-PE, CD28-BV785, CD28-PerCP-Cy5.5, CD70-PE, CD127--BV711, CD69-BV650, and granzyme B) were purchased from Biolegend, and antibodies against CD8-BUV496, CD27-BUV395, and perforin-PE-CF594 were purchased from BD.

Lu J., Chen G., Sorokina A., Nguyen T., Wallace T., Nguyen C., Dunn C., Wang S., Ellis S., Shi G., McKelvey J., Sharov A., Liu Y.T., Schneck J, & Weng N.P. (2022). Cytomegalovirus infection reduced CD70 expression, signaling and expansion of viral specific memory CD8+ T cells in healthy human adults. Immunity & Ageing : I & A, 19, 54.

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Comprehensive Profiling of T Cell Markers

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Cell surface proteins on (CAR-)T cells were stained with the following antibodies: annexin V-PE, CCR7-PerCP/Cy5.5, CD3-allophycocyanin (APC), CD3-BV421, CD4-APC/H7, CD8a-PE/Cy7, CD25-PE/Cy7, CD69-fluorescein isothiocyanate (FITC), CD95-PE, CD127-PerCP/Cy5.5, CXCR3-AF488 (all from BD Biosciences, San Jose, CA); CD8a-APC, CD8a-FITC, CD27-PE, CD28-FITC, CD45RA-APC, LAG-3-PE, TIGIT-PerCP/Cy5.5, TIM-3-FITC (all from BioLegend, San Diego, CA); and PD-1-APC (eBioscience, Thermo Fisher Scientific). Monocytes were stained with CD14-PE (eBioscience). To determine CAR expression, cells were labeled with a recombinant Fc-tagged BCMA protein (Enzo Life Sciences, Farmingdale, NY) followed by a BV421-conjugated anti-Fc antibody (BioLegend). Samples prepared from mouse organs were treated with Fc block (BD Biosciences) prior to staining. Cells were washed and resuspended in fluorescence-activated cell sorting (FACS) buffer (1% FCS in PBS) or 1% paraformaldehyde prior to acquisition. In experiments to assess viability and/or apoptosis, cells were resuspended in FACS buffer containing 1 μg/mL 7-aminoactinomycin D (7-AAD). In all other experiments, live cells were gated based on forward and side scatter.
All flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software version X.0.7 (Treestar, Ashland, OR).

Battram A.M., Oliver-Caldés A., Suárez-Lledó M., Lozano M., Bosch i Crespo M., Martínez-Cibrián N., Cid J., Moreno D.F., Rodríguez-Lobato L.G., Urbano-Ispizua A, & Fernández de Larrea C. (2022). T cells isolated from G-CSF-treated multiple myeloma patients are suitable for the generation of BCMA-directed CAR-T cells. Molecular Therapy. Methods & Clinical Development, 26, 207-223.

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Multi-parameter Flow Cytometry Analysis

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Surface staining was performed on cells washed with FACS buffer (PBS, 2% FCS, 0.05% sodium azide, 0.5 mM EDTA). Dead cells were detected with Zombie NIR™ Fixable Viability Kit (Biolegend) according to the manufacturer’s protocol. Cells were then stained for 20 minutes at room temperature with antibody mixes including the following: CD3-PeCy7, CD3-PerCPCy5.5, CD4-FITC, CD4-PECy7, CD8-PerCPCy5.5, CD14-PerCPCy5.5, CD16-FITC, CD45RO-PE, CD45RA-APC, CCR6-PE, CXCR3-APC, and HLA-DR-PECy7 (all from Biolegend). For intracellular staining, cells were washed after surface staining, fixed with 2% PFA for 15 minutes at RT, and washed with permeabilization buffer (FoxP3 eBioscience buffer). Cells were then intracellular stained for 30 minutes at RT with antibody mixes that were made in permeabilization buffer, including interferon (IFN)γ-PB and IL-4-AF647 (all from Biolegend). Cells were washed with FACS buffer and acquired using a BD Canto II. All experiments were analysed using FlowJo v10.4 (Becton, Dickinson and Company, Ashland, OR, USA).

Waller K.J., Saihi H., Li W., Brindley J.H., De Jong A., Syn W.K., Bessant C, & Alazawi W. (2022). Single-cell phenotypes of peripheral blood immune cells in early and late stages of non-alcoholic fatty liver disease. Clinical and Molecular Hepatology, 29(2), 417-432.

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IL-2 Stimulation of CD4+ T Cells

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CD4+ T cells (0.2×10⁶ cells) were stimulated in complete medium in 96-well plates with recombinant IL-2 (Peprotech) for 0 to 180 minutes. At the end of stimulation, cells were washed, stained for dead cells using the Zombie Aqua Fixable Viability Kit (20 min, 4°C) (Biolegend), washed, permeabilized and stained with CD45RA APC (Biolegend) and phospho-STAT5 Alexa Fluor 488 (Cell Signaling Technologies) using the Phospho-Epitopes Exposure kit (PerFix EXPOSE, Beckman Coulter) and following the manufacturer’s instructions.

Comte D., Karampetsou M.P., Kis-Toth K., Yoshida N., Bradley S.J., Kyttaris V.C, & Tsokos G.C. (2017). CD4+ T cells from SLE patients respond poorly to exogenous IL-2. Arthritis & rheumatology (Hoboken, N.J.), 69(4), 808-813.

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CAR T Cell Phenotypic Characterization

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Eight days after activation, CAR T cells were either maintained alone or cocultured with SKBR3 tumor cells for 8 and 24 h under control and pro-oxidative conditions. Then, CAR T cells were collected, fixed, and stained to assess: i) CAR construct expression, ii) exhaustion state, and iii) memory state. The following antibodies were used for staining: i) CAR construct expression, IgG-PE (Southern Biotech, 2042-09); ii) exhaustion state CD3-PE (BD Biosciences, 345765), CD4-V450 (BD Biosciences Biosciences, 560345), CD8-FITC (BD Biosciences, 555366), PD1-APC (BioLegend, 367406), TIM3-APC-Cy7 (BioLegend, 345026), and LAG3-PE-Cy7 (BioLegend, 369310); and iii) memory state CD4-AF700 (BioLegend, 317426), CD8-APC-Cy7 (BioLegend, 344714), CD45RA-APC (BioLegend, 304112), CD45RO-PerCP-Cy5.5 (BioLegend, 304222), CCR7-PE-Cy7 (BioLegend, G043H7), CD62L-PE-Cy5 (BioLegend, 304808), Granzyme B-FITC (BD, 558132), IFN-γ-BV621 (BioLegend, 502536), CD127-BV421(BioLegend, 351310), and CD57-PE (BioLegend, 359612) according to the manufacturer’s instructions.

Balta E., Janzen N., Kirchgessner H., Toufaki V., Orlik C., Liang J., Lairikyengbam D., Abken H., Niesler B., Müller-Decker K., Ruppert T, & Samstag Y. (2022). Expression of TRX1 optimizes the antitumor functions of human CAR T cells and confers resistance to a pro-oxidative tumor microenvironment. Frontiers in Immunology, 13, 1063313.

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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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Comprehensive CAR and Immunophenotyping Analysis

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FACS analysis of cell surface CAR and protein expression was performed using an LSR II Fortessa flow cytometer (BD Biosciences). CD22-CAR was detected by incubation with 22-Fc (R&D Systems), followed by incubation with human IgG-specific PE-F(ab)2 (Thermo Fisher Scientific). The following human monoclonal antibodies were used for detection of cell surface proteins: CD22-APC, CD22-PE, CD19-Pacific Blue, CD45-PerCP/Cy5.5, CD3-APC/Cy7, PD1-PE/Cy7, LAG3-APC, TIM3-Pacific Blue, CD8-APC, CD8-PE/Cy7, CD45RA-APC, CD45RO-PE/Cy7, CCR7-Pacific Blue, CD4-Pacific Blue, CD69-APC (all from BioLegend). CD22 site density was determined using QuantiBrite-PE beads (BD Biosciences) using methods previously described (PMID: 20872890). Dead cells were identified using eFluor 506 fixable viability dye (Thermo Fisher Scientific). GFP-expressing leukemia was identified through the FITC channel.

Ramakrishna S., Highfill S.L., Walsh Z., Nguyen S.M., Lei H., Shern J.F., Qin H., Kraft I., Stetler-Stevenson M., Yuan C.M., Hwang J., Feng Y., Zhu Z., Dimitrov D., Shah N.N, & Fry T.J. (2019). Modulation of Target Antigen Density Improves CAR T Cell Functionality and Persistence. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(17), 5329-5341.

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