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Cd45ra hi100

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CD45RA (HI100) is a cell surface marker antibody that recognizes the CD45RA isoform of the CD45 protein. CD45 is a tyrosine phosphatase that is expressed on the surface of most hematopoietic cells. The CD45RA isoform is primarily found on naive T cells and some B cells. This product can be used for the identification and enumeration of CD45RA-positive cell populations in flow cytometry applications.

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Lab products found in correlation

Lsrfortessa, by BD (2 mentions) Mouse cd45 clone 30 f11, by BioLegend (2 mentions) Cd3 clone okt3, by BioLegend (2 mentions) Cd8 clone sk1, by BioLegend (2 mentions) Cd45ro clone uchl1, by BioLegend (2 mentions) Human cd45 clone hi30, by BioLegend (2 mentions) Cd4 clone a161a1, by BioLegend (2 mentions) Pd 1 clone mih4, by BD (2 mentions) Zombie yellow fixable viability kit, by BioLegend (2 mentions) Facsaria 2, by BD (2 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions) Live dead fixable dead cell stain, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Facsymphony, by BD (2 mentions) Granzyme b gb11, by BioLegend (2 mentions) Cd8 sk1 or rpa t8, by BioLegend (2 mentions) Pd 1 eh12.1, by BioLegend (2 mentions) Ccr7 g043h7, by BioLegend (2 mentions) Cd4 gk1, by BioLegend (1 mentions) Cd45ro uchl1, by BioLegend (1 mentions)

Close spelling variants of this product

CD45RA APC (clone HI100, (5 citations) Anti-CD45RA-APC-Cy7 (HI100), (4 citations) Anti-CD45RA (clone HI100), (3 citations) CD45RA HI100 APC-CY7 304128 (3 citations) CD45RA PerCP-Cy5.5 (HI100), (3 citations) CD45RA (clone HI100; (3 citations) ) PE-Cy7 anti-CD45RA (clone HI100, (2 citations) CD45RA-BV510 (clone HI100), (2 citations) CD45RA V500 (clone HI100; (2 citations) CD45RA/Alexa Fluor 700 (HI100; (2 citations)

10 protocols using cd45ra hi100

1

T Cell Phenotyping Protocol

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All samples were analyzed with an LSR Fortessa (BD Bioscience), and data were analyzed using FlowJo software (FlowJo). T cell phenotype was evaluated via Zombie Yellow Fixable Viability Kit (BioLegend), human CD45 (clone HI30; BioLegend), mouse CD45 (clone 30F11; BioLegend), CD3 (clone OKT3; BioLegend), CD4 (clone A161A1; BioLegend), CD8 (clone SK1; BioLegend), PD-1 (clone MIH4; BD Bioscience), CD45RO (clone UCHL1; BioLegend), and CD45RA (HI100; BioLegend).
Wang Y., Buck A., Grimaud M., Culhane A.C., Kodangattil S., Razimbaud C., Bonal D.M., Nguyen Q.D., Zhu Z., Wei K., O'Donnell M.L., Huang Y., Signoretti S., Choueiri T.K., Freeman G.J., Zhu Q, & Marasco W.A. (2021). Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model. Molecular Therapy Oncolytics, 24, 385-399.
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2

Comprehensive Immune Cell Profiling

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PBMCs were stained with CD197 (G034H7) at 37°C for 20’, followed by CD4 (RPA-T4), CD8 (RPA-T8), CD69 (FN50), CD95 (DX2), CD45RA (HI100), and CD45RO (UCHL1) for 30’ at 4°C (Biolegend, San Diego, CA). Murine cells were stained with CD4 (GK1.5), CD8a (53–5.8), CD25 (PC61), CD44 (IM7), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), GITR (DTA-1), Sca-1 (D7), TCRβ (H57–597), TNF-ɑ (MP6-XT22) (all Biolegend) and live-dead (Life Technologies; Carlsbad, CA). Cells were fixed and permeabilized according to instructions (ThermoFisher). Human PBMCs were stained with IFN-ɣ (B27), Ki67 (Ki67), and TNF-ɑ (Mab11). Murine cells were stained with Bcl-2 (BCL-10C4) and TNF-ɑ (Mab11). Samples were acquired on an LSR-II (BD; San Diego, CA), and data were analyzed using FlowJo v10.3.1 (TreeStar; Ashland, OR).
Padgett L.E., Dinh H.Q., Wu R., Gaddis D.E., Araujo D.J., Winkels H., Nguyen A., McNamara C.A, & Hedrick C.C. (2020). Naive CD8+ T Cells Expressing CD95 Increase Human Cardiovascular Disease Severity. Arteriosclerosis, thrombosis, and vascular biology, 40(12), 2845-2859.
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3

Flow Cytometric Analysis of T Cell Phenotypes

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To evaluate the expression of T cell surface markers by flow cytometry, we incubated tissue and blood cell suspensions with Human TruStain FcX (BioLegend) and stained with following fluorochrome-conjugated antibodies: CD3 (UCHT1, BD Biosciences; OKT3, BioLegend), CD4 (SK3, BD Biosciences; SK3, Tonbo Biosciences), CD8 (SK1, BioLegend; RPA-T8, BD Biosciences), CCR7 (G043H7; BioLegend), CD45RA (HI100; BioLegend), CD25 (BC96; BioLegend), CD127 (A019D5; BioLegend), CD69 (FN50; BioLegend), CD103 (Ber-ACT8; BioLegend), CD45 (HI30; BioLegend), and Fixable Viability Dye eFluor 780 (eBioscience). For stimulation/proliferation assays, we magnetically enriched for CD3+ T cells from single cell suspensions, stained cells with Cell Proliferation Dye eFluor 450 (eBioscience), and cultured cells for up to 120 h with or without TCR stimulation as above. At indicated time points, we performed intercellular staining of NME1 (11615-H07E; Sino Biological) using a Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences) for fixation and permeabilization of cells according to manufacturer’s instructions. We acquired cell fluorescence data using a BD LSR II flow cytometer and used FCS Express (De Novo Software) for analysis. The results are summarized in Supplementary Fig. 2 and the gating strategy is shown in Supplementary Fig. 18a.
Szabo P.A., Levitin H.M., Miron M., Snyder M.E., Senda T., Yuan J., Cheng Y.L., Bush E.C., Dogra P., Thapa P., Farber D.L, & Sims P.A. (2019). Single-cell transcriptomics of human T cells reveals tissue and activation signatures in health and disease. Nature Communications, 10, 4706.
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4

Comprehensive PBMC Phenotypic Analysis

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Phenotypic analysis was performed on PBMCs using panels containing Live/Dead Blue (Life technologies), CD1c (AD5-8E7; Miltenyi), CD3 (SP34-2 and SK7; BD), CD10 (HI10a; Biolegend), CD11c (B-Ly6, BD), CD14 (M5E2, BD), CD16 (3GE, Biolegend), CD19 (HIB19; Biolegend), CD20 (L27, BD), CD34 (561, Biolegend), CD45RA (HI100, Biolegend), CD56 (HCD56, BD), CD62L (SK11, BD), CD66abce (TET2, Miltenyi Biotec), CD80 (L307.4, BD), CD86 (2331; BD); CD123 (7G3; BD), CD133 (7, Biolegend), CD141 (AD5-14H12; Miltenyi), CD146 (P1H12; Biolegend), HLA-DR (TU36, Life technologies), CCR2 (K036C2, Biolegend), CCR4 (L291H4, Biolegend), CCR6 (11A9, BD) and CCR7 (150503, BD) (Panels summarized in S3 Table). Fixation was performed with 1% paraformaldehyde. Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).
Vangeti S., Strandin T., Liu S., Tauriainen J., Räisänen-Sokolowski A., Cabrera L., Hassinen A., Mäkelä S., Mustonen J., Vaheri A., Vapalahti O., Klingström J, & Smed-Sörensen A. (2021). Monocyte subset redistribution from blood to kidneys in patients with Puumala virus caused hemorrhagic fever with renal syndrome. PLoS Pathogens, 17(3), e1009400.
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5

Multiparameter Flow Cytometry Analysis

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Single-cell suspensions were washed with PBS and stained with LIVE/DEAD Fixable Dead Cell Stain (Thermo Fisher) for 15 minutes, followed by staining with an optimized antibody cocktail for 20 minutes at room temperature (RT). Cells were then washed with FACS buffer (PBS containing 2% FBS) and fixed in PBS containing 1% paraformaldehyde (Sigma-Aldrich). For samples requiring intracellular staining, cells were processed using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher) according to the manufacture’s instruction. Samples were acquired using a FACSymphony (BD Biosciences). Data were analyzed with FlowJo software (version 9.8.8 or higher, BD Biosciences). All sorting experiments were carried out on freshly isolated cells using a FACSAriaII (BD Biosciences). Antibodies used include: CD45 (HI30), CD103 (Ber-ACT8), CD45RA (HI100, Biolegend), CD3 (UCHT1), CD69 (FN50), CD8 (SK1 or RPA-T8), CCR7 (G043H7, Biolegend), CD25 (M-A251), CD127 (HIL-7R-M21), CD4 (RPA-T4), CCR5 (2D7), PD-1 (EH12.1, Biolegend), Granzyme B (GB11), Foxp3 (259D/C7), CTLA4 (BNI3), IL-17A (N49-653), IL-2 (5344.111), (TNFα (Mab11), IFNγ (B27). All antibodies were from BD Biosciences unless otherwise noted.
Davis A.S., Vick S.C., Pattacini L., Voillet V., Hughes S.M., Lentz G.M., Kirby A.C., Fialkow M.F., Gottardo R., Hladik F., Lund J.M, & Prlic M. (2021). The human memory T cell compartment changes across tissues of the female reproductive tract. Mucosal immunology, 14(4), 862-872.
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6

Multiparametric Flow Cytometry Analysis

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The following antibodies were used for flowcytometry: CD3 (HIT3a, Becton-Dickinson (BD)), CD4 (SK3, BD), CD45RA (HI100, Biolegend), CXCR5 (TG2/CXCR5,Biolegend and RF8B2,BD), CCR7 (3D12, BD), PD-1 (NAT), CD25 (2A3, BD), CD127 (A7R34, eBioscience), CRTAM(R&D), Granzyme A (GB11,BD), Foxp3 (259D/C7, BD), CD57(HNK-1,BD), IL-17 (SCPL1362, BD), IL-10 (JES3-19F1,BD), IFN-g (25723.11,BD), IL-4 (7A3-3,BD), IL-21 (3A3-N2.1, BD), STAT3(4/P-STAT3,BD), and cell trace violet (CTV,Life Technologies). Data were acquired on FACSCaliber or FACSCanto II and data were then analyzed with FlowJo, version (8.7) (Treestar).
Alshekaili J., Chand R., Lee C.E., Corley S., Kwong K., Papa I., Fulcher D.A., Randall K.L., Leiding J.W., Ma C.S., Wilkins M.R., Uzel G., Goodnow C.C., Vinuesa C.G., Tangye S.G, & Cook M.C. (2018). STAT3 regulates cytotoxicity of human CD57+ CD4+ T cells in blood and lymphoid follicles. Scientific Reports, 8, 3529.
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7

Comprehensive T cell Phenotyping Protocol

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All samples were analyzed with an LSR Fortessa (BD Bioscience) and data were analyzed using FlowJo software (FlowJo LLC). T cell phenotype was evaluated via:Zombie Yellow Fixable Viability Kit (BioLegend), human CD45 (clone HI30, BioLegend), mouse CD45 (clone 30F11, BioLegend), CD3 (clone OKT3, BioLegend), CD4 (clone A161A1, BioLegend), CD8 (clone SK1, BioLegend), CD19 (clone HIB19, BioLegend), CD27 (clone M-T271, BioLegend), CD62L (DREG-56, BioLegend), PD-1 (clone MIH4, BD Bioscience), TIM-3 (clone F38-2E2, BioLegend), CD45RO (clone UCHL1, BioLegend), CD45RA (HI100, BioLegend).
Wang Y., Cho J.W., Kastrunes G., Buck A., Razimbaud C., Culhane A.C., Sun J., Braun D.A., Choueiri T.K., Wu C.J., Jones K., Nguyen Q.D., Zhu Z., Wei K., Zhu Q., Signoretti S., Freeman G.J., Hemberg M, & Marasco W.A. (2024). Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model. iScience, 27(2), 108879.
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8

Multiparameter Flow Cytometry Analysis

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Single-cell suspensions were washed with PBS and stained with LIVE/DEAD Fixable Dead Cell Stain (Thermo Fisher) for 15 minutes, followed by staining with an optimized antibody cocktail for 20 minutes at room temperature (RT). Cells were then washed with FACS buffer (PBS containing 2% FBS) and fixed in PBS containing 1% paraformaldehyde (Sigma-Aldrich). For samples requiring intracellular staining, cells were processed using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher) according to the manufacture’s instruction. Samples were acquired using a FACSymphony (BD Biosciences). Data were analyzed with FlowJo software (version 9.8.8 or higher, BD Biosciences). All sorting experiments were carried out on freshly isolated cells using a FACSAriaII (BD Biosciences). Antibodies used include: CD45 (HI30), CD103 (Ber-ACT8), CD45RA (HI100, Biolegend), CD3 (UCHT1), CD69 (FN50), CD8 (SK1 or RPA-T8), CCR7 (G043H7, Biolegend), CD25 (M-A251), CD127 (HIL-7R-M21), CD4 (RPA-T4), CCR5 (2D7), PD-1 (EH12.1, Biolegend), Granzyme B (GB11), Foxp3 (259D/C7), CTLA4 (BNI3), IL-17A (N49-653), IL-2 (5344.111), (TNFα (Mab11), IFNγ (B27). All antibodies were from BD Biosciences unless otherwise noted.
Davis A.S., Vick S.C., Pattacini L., Voillet V., Hughes S.M., Lentz G.M., Kirby A.C., Fialkow M.F., Gottardo R., Hladik F., Lund J.M, & Prlic M. (2021). The human memory T cell compartment changes across tissues of the female reproductive tract. Mucosal immunology, 14(4), 862-872.
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9

Characterization of Regulatory T Cells

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Human-specific monoclonal antibodies, including CD4 (A161A1), CD25 (BC96), Foxp3 (206D), and CD45RA (HI100), were purchased from BioLegend (UK), and CD127 (A019D5) was purchased from BD Pharmingen (USA). Foxp3 (206D) was used for intracellular flow cytometry assays, while the others were used for surface staining. A MACSQuant Analyzer 10 (Miltenyi Biotec, Germany) was used for detection, and results were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
Zhou J., Shao Q., Lu Y., Li Y., Xu Z., Zhou B., Chen Q., Li X., Xu X., Pan Y., Deng Z., Wang Y., Yu Y, & Gu J. (2022). Monocarboxylate transporter upregulation in induced regulatory T cells promotes resistance to anti-PD-1 therapy in hepatocellular carcinoma patients. Frontiers in Oncology, 12, 960066.
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10

Flow Cytometry Analysis of Immune Cells

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We applied established protocols as previously described (5 (link)). The following antibodies were used for flow cytometry analysis; For mouse, CD4 (GK1.5), CD45 (30-F11), CD90.2 (53–2.1), and IL-17A (JC11–18H10.1) were purchased from BioLegend. For human, CD45RA (HI100) was purchased from BioLegend. A CD4 (SK3) was purchased from eBioscience. A Zombie Aqua™ Fixable Viability Kit (intracellular) staining was performed for eliminating dead cells. Surface staining was performed on ice for 20–30 min. Absolute cell numbers were calculated on the basis of the percentage of each cell population. For intracellular staining, harvested cells were stimulated for 4 hours in culture medium with PMA (Sigma-Aldrich), ionomycin (Sigma-Aldrich), and monensin (BD Biosciences). Cytofix/Cytoperm and Perm/Wash buffer (IL-17A/ IFNγ; BD Biosciences) was used for fixation and permeabilization. All flow cytometry data were acquired on a BD LSRII (BD Biosciences) or Cytoflex LX (Beckman Coulter) and analyzed with FlowJo (FlowJo, llc). All procedures were performed according to the manufacturer’s instructions.
Kono M., Yoshida N., Maeda K., Suárez-Fueyo A., Kyttaris V.C, & Tsokos G.C. (2019). Glutaminase 1 Inhibition Reduces Glycolysis and Ameliorates Lupus-like Disease in MRL/lpr Mice and Experimental Autoimmune Encephalomyelitis. Arthritis & rheumatology (Hoboken, N.J.), 71(11), 1869-1878.
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