Ultra-LEAF(TM) Purified anti-mouse CD3epsilon 100360 from BioLegend

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eLife
November 23 2020

Fig 1: Respiratory restriction has only marginal effect on cytoplasmic function during early activation.(A) Heatmap showing relative amounts of key energy-related metabolites (as indicated in the figure) extracted from CD8+ T cells activated for 5 hr using anti-CD3/28 (T-Early) oligomycin treated or untreated (Ctrl). (B) Same as in A, extracted from CD8+ T cells activated for 24 hr using anti-CD3/28 (T-Late). (C) Splenocytes were stimulated using anti-CD3/28 for 9 hr (T-Early), 12 hr (T-Late) or left untreated (Naïve- gray). Cells were then treated with 300 nM oligomycin or left untreated for 1 hr. Protein extract from isolated CD8+ T cells from all samples were then subjected for immunoblot analysis using anti p-AMPKa or anti AMPKa. (n = 3 experiments). (D-G) CellTrace-labeled splenocytes from WT or LCK-cre/Slc25a5floxp (ANT2ko) mice were stimulated using anti-CD3/CD28, with or without 60 nM oligomycin. Seventy-two hours post activation cells were analyzed by flow cytometry (n = 5 biological replicates). (D) Representative Flow cytometry plots of FSC vs. CD25 gated on CD8+ T cells from wild-type mice (WT- left) or ANT2ko mice untreated or treated with 60 nM oligomycin (middle and right panels respectively). Numbers indicate the frequencies of CD25+ cells. (E) Bar graph summarizes results in D. (P value ** 0.0079). (F) Representative Flow cytometry overlay histogram of CellTrace intensity gated on CD8+ T cells from either WT cells (front) or ANTko cells that were untreated (middle) or treated with oligomycin (back). (G) Bar graph summarizing the results in F as proliferation index. (P value ** 0.0079). (H-K) CellTrace-labeled splenocytes from WT mice were stimulated using anti-CD3/CD28, in the presence of the indicated concentrations of the pan-ANT inhibitor, bongkrekic acid. Seventy-two hours post activation cells were analyzed by flow cytometry analysis. (n = 5 biological replicates). (H) Representative Flow cytometry plots of FSC vs. CD25 gated on CD8+ T cells from WT splenocytes untreated or treated with the indicated concentrations of bongkrekic acid. Numbers indicate the frequencies of CD25+ cells. (I) Bar graph summarizing the results in H. (J) Representative Flow cytometry overlay histogram of CellTrace intensity gated on CD8+ T cells that were either untreated or treated with the indicated concentrations of bongkrekic acid. (K) Bar graph summarizing the results in J as proliferation index. Statistical method, non-parametric Mann–Whitney test, mean ± s.e.m.
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Nature communications
November 24 2021

Fig 2: LSD1/CoREST complex physically interacts with TCF1 and antagonizes its transcriptional activity.a MFI of TCF1 in CD8+TCF1+PD-1int TILs analyzed by flow cytometry (n = 4 per group). b The physical interaction between LSD1 and TCF1 isoforms as well as other proteins was examined by co-immunoprecipitation (co-IP) assays using whole-cell extract (WCE) of HEK293T cells transfected with FLAG-HA-tagged LSD1. c Schematic representation of different isoforms of mouse TCF1. d Co-IP assays precipitating HA-tagged TCF1 isoforms and detecting LSD1 in eluted IP fractions. e Co-IP assays precipitating Flag-tagged TCF1 (referring to p45 unless otherwise specified) and detecting components of the LSD1/CoREST complex in eluted IP fractions. f–j TCF/LEF reporter assay measuring relative luciferase units (RLU) in response to transfection with plasmids expressing indicated proteins (f–j, n = 4 per condition) or shRNA targeting LSD1 (i) and the treatment with iCRT3 (i) (n = 4 per condition). Protein expression for plasmid transfection in f is shown by immunoblotting (g). k, l Representative flow plots (k) and Slamf6 MFI (l) of splenic CD8+ T cells activated in vitro for 3 days with anti-CD3/anti-CD28 in the presence of vehicle, GSK2879552 or iCRT3 (n = 3 per treatment). Data represent two (a, b, d–j) or three (k, l) independent experiments and are presented as mean ± SEM (a) or mean ± SD (f, h–j, l). Statistical significance was determined by two-sided unpaired t test (a, f, h–j, l). Source data are provided as a Source data file.
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Neurobiology of Pain
August 01 2022

Fig 3: LD-IL-2 treatment increases the production of IL-10 and TGFß1 in splenocytes. (A) The amount of IL-10 in culture supernatant in response to anti-CD3 and anti-CD28 stimulation measured by ELISA (n = 5 and 7 in saline and LD-IL-2 groups, respectively). *p < 0.05, two-tailed t test. (B) Gating strategy for individual cell populations. (C-E) The frequencies of TGFß1+ cells (C), TGFß1+EGFP+ cells (D), and TGFß1+EGFP- cells (E) among CD4+ splenocytes from saline- and ld-IL-2-treated DEREG mice (n = 10 and 12 in saline and LD-IL-2 groups, respectively). *p < 0.05, two-tailed t test. (F-G) The number (F) and the frequency (G) of CD4+ cells in total splenocytes in R1 (gated for lymphocytes) from saline- and LD-IL-2-treated DEREG mice (same as in B-D).
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Nature communications
January 11 2022

Fig 4: Anti-BTNL2 mAb treatment decreases the tumour infiltration of ?dT17 cells.a, b After isotype control Ab or anti-BTNL2 mAb treatment (200 µg/mouse), infiltrated live CD3 + ?d T lymphocytes which producing IL-17A or IFN-? in subcutaneous CT26 (a) and A20 (b) tumours were analyzed by flow cytometry as indicated (a, n = 15, P = 0.014 for ?dT+IL-17A+ cell Percentages, NS for ?dT+IFN-?+ cell Percentages and b, n = 14, P < 0.0001 for ?dT+IL-17A+ cell Percentages, NS for ?dT+IFN-?+ cell Percentages). Serum IL-17A was examined by ELISA (n = 15, P < 0.0001 for a). c Splenocytes were cultured in the presence of plate-coated Fc, WT-BTNL2-Fc or N4S-BTNL2-Fc recombinant proteins (10 µg/ml) for 48 h, followed by flow cytometry analysis of ?dT17 and Th17 (cells were restimulated with Cell Activation Cocktail (with Brefeldin A) for 4 h, and were gated by live CD45+, n = 9, P < 0.0001 for Fc vs WT-BTNL2-Fc ?dT+IL-17A+ cell Percentages, P < 0.0001 for Fc vs N4S-BTNL2-Fc ?dT+IL-17A+ cell Percentages, P = 0.003 for WT-BTNL2-Fc vs N4S-BTNL2-Fc ?dT+IL-17A+ cell Percentages, NS for CD4+IL-17A+ cell Percentages). d FACS sorted ?d T cells were cultured in the presence of plate-coated Fc, WT-BTNL2-Fc or N4S-BTNL2-Fc with or without IL-1ß and IL-23 for 24 h. ELISA was performed to analyze IL-17A production (n = 7, P = 0.0003 for Fc vs WT-BTNL2-Fc, P < 0.0001 for Fc vs N4S-BTNL2-Fc, P = 0.0456 for IL-1ß + IL-23+Fc vs IL-1ß + IL-23+WT-BTNL2-Fc, P = 0.0098 for IL-1ß + IL-23+Fc vs IL-1ß + IL-23 + N4S-BTNL2-Fc). e FACS sorted ?d T cells were cultured in the presence of plate-coated Fc, WT-BTNL2-Fc or N4S-BTNL2-Fc together with IL-1ß and IL-23 for 24 h, followed by real-time PCR analysis of RORC expression (n = 10, P = 0.0184 for Fc vs WT-BTNL2-Fc, P < 0.0001 for Fc vs N4S-BTNL2-Fc). f IL-17A were neutralized by neutralizing antibody described in the Methods (100 µg/mouse), and CT26 tumour growth kinetics was shown (n = 16 for each group, P = 0.018). g Primary CT26 tumour growth kinetics of mice after intraperitoneal injection of control Ab or anti-BTNL2 mAb (200 µg/mouse) together with Fc or IL-17A-Fc recombinant proteins was shown. (n = 15 for each group, P < 0.0001). Fc or IL-17A-Fc recombinant proteins (5 µg/mouse) were intraperitoneal injected at day 1, 4, 7, 10 and 13 after tumour implantation. Right panel indicates the purified Fc and IL-17A-Fc recombinant proteins analyzed by western blot. All data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on Mann–Whitney test for (a, b), one-way ANOVA for (c–e) and Two-way ANOVA for (f, g). Data are representative of three independent experiments.
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Journal for immunotherapy of cancer
August 01 2022

Fig 5: 90Y-NM600 increased CD8 +T cell infiltration into prostate tumors but also increased checkpoint molecule expression. TRAMP-C1 tumors were collected from mice (n=5 per group) at several time points following no treatment (control), low-dose TRT, or high-dose TRT, digested, and evaluated for cellular composition by flow cytometry. Shown are: (A) CD3 +CD8+T cells as a percentage of CD45 +cells. (B) CD69 (left) and CD137 (right) median fluorescence intensity (MFI) on CD8 +T cells. (C) Percent of CD8 +T cells bearing markers for effector memory (CD44 +CD27-CD62L-), central memory (CD44 +CD27+CD62L+), resident memory (CD44 +CD69+CD103+) or short-lived effector (KLRG-1 +CD127-) cell phenotypes. (D) MFI of PD-1, CTLA-4, LAG-3, and VISTA on CD8 +T cells (E) PD-L1 MFI on dendritic cells (CD11b-CD11c+MHCII+ cells), myeloid-derived suppressor cells (MDSCs; CD11b+Gr-1+), and tumor cells (CD45-). *P<0.05, **p<0.01, *** p<0.001 compared with untreated controls via two-way ANOVA with Benjamini-Hochberg adjustment of pairwise tests. To perform pairwise testing, untreated controls were pooled then compared with each TRT-treated group. Error bars represent mean±SE. Results shown are from one experiment and are representative of two independent experiments. ANOVA, analysis of variance; DCs, dendritic cells; TRT, targeted radionuclide therapy.

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Ultra-LEAF(TM) Purified anti-mouse CD3epsilon

The Ultra-LEAF(TM) Purified anti-mouse CD3epsilon Antibody from BioLegend is a Hamster Monoclonal antibody to CD3E. This antibody recognizes Mouse antigen. The Ultra-LEAF(TM) Purified anti-mouse CD3epsilon Antibody has been validated for the following applications: Block/Neutralize/Inhibit, Flow Cytometry, Immunocytochemistry, Immunohistochemistry, Immunohistochemistry - fixed, Immunoprecipitation, Western Blot, and Other.

Description

Ultra-LEAF(TM) Purified anti-mouse CD3epsilon [145-2C11]

Product Specs
ItemUltra-LEAF(TM) Purified anti-mouse CD3epsilon
CompanyBioLegend
Price Pricing Info Supplier Page View Company Product Page
Catalog Number100360
Quantity25 mg
ApplicationsFlow Cytometry (FC), Immunohistochemistry (IHC)-F, Immunoprecipitation (IP), Activation, Block, Western Blot (WB), Immunocytochemistry (ICC)
TargetCD3epsilon
ReactivityMouse (Ms)
ClonalityMonoclonal
NCBI Gene AliasesCD3, CD3epsilon, T3e
NCBI Full Gene NameCD3 antigen, epsilon polypeptide
ImmunogenH-2K^b-specific mouse cytotoxic T lymphocyte clone BM10-37
Antibody TypePrimary
HostArmenian Hamster
Clone145-2C11
IsotypeIgG
Format0.2 µm filtered in phosphate-buffered solution, pH 7.2, containing no preservative
Conjugate/TagUnconjugated, Ultra Low Endotoxin Azide Free

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(1) Macrophages in the synovial lining niche initiate neutrophil recruitment and articular inflammationJournal of Experimental MedicineAugust 7, 2023Kristina Zec, Barbora Schonfeldova, Zhichao Ai, Erinke Van Grinsven, Gabriela Pirgova, Hayley L. Eames, Dorothée L. Berthold, Moustafa Attar, Ewoud B. Compeer, Tal I. Arnon, et al.supplemented with a-CD28 at 2 μg/ml (102116, clone 37.51; BioLegend) in 24-well plates precoated with a-CD3 (100340, clone 145-2C11; BioLegend). On day 2 after activation, cells were split at 1:3 to 1:5 in a fresh 24-well

(2) Distinct bidirectional regulation of LFA1 and α4β7 by Rap1 and integrin adaptors in T cells under shear flowCell reportsJune 27, 2023Kamioka Y, Ueda Y, Kondo N, Tokuhiro K, Ikeda Y, Bergmeier W, Kinashi T.AB_313692 Purified anti-mouse CD28,Biolegend,Cat# 102116; RRID: AB_11147170 Purified anti-mouse CD3ϵ,Biolegend,Cat# 100340; RRID: AB_11149115 Purified anti-mouse MAdCAM-1 (MECA-89) from hybridoma,ATCC/This paper,Cat# HB-292 Purified

(3) ERK and USP5 govern PD-1 homeostasis via deubiquitination to modulate tumor immunotherapyNature communicationsMay 19, 2023Xiao X, Shi J, He C, Bu X, Sun Y, Gao M, Xiang B, Xiong W, Dai P, Mao Q, et al.selection magnetic beads (19853, Stemcell Technologies) were stimulated with plate-bound anti-CD3 antibody (100340, Biolegend) and anti-CD28 antibodies (102116, Biolegend) for 48 h. CD8+ T cells were then cultured in

(4) Piceatannol induces regulatory T cells and modulates the inflammatory response and adipogenesisBiomedicine & PharmacotherapyMay 1, 2023Ahmed Rakib, Mousumi Mandal, Anaum Showkat, Sonia Kiran, Soumi Mazumdar, Bhupesh Singla, Aman Bajwa, Santosh Kumar, Frank Park, Udai P. Singhindividual 6-well plates were coated with anti-CD3 antibody (5 μg/mL; clone: 145–2C11, Catalog No. 100360, BioLegend, San Diego, CA) and kept overnight at 4°C. In the experiment, single-cell suspensions of

(5) ITK signaling regulates a switch between T helper 17 and T regulatory cell lineages via a calcium-mediated pathwaybioRxiv : the preprint server for biologyApril 3, 2023Anannya O, Huang W, August A.(29F.1A12; 135224), CD4 (GK1.5; 100427), CD45.1 (A20; 110716), CD45.2 (104; 109819), CD3e (145-2C11; 100340) and CD28 (35.51; 102112) were purchased from Biolegend (San Diego, CA). Antibody for S6 (pS235/pS236)

(6) Itaconate ameliorates autoimmunity by modulating T cell imbalance via metabolic and epigenetic reprogrammingNature communicationsFebruary 27, 2023Aso K, Kono M, Kanda M, Kudo Y, Sakiyama K, Hisada R, Karino K, Ueda Y, Nakazawa D, Fujieda Y, et al.(MP Biomedicals) and stimulated for 2–3 days with anti-CD3 (0.25 μg mL−1, clone 145-2C11, Biolegend, 100340) and anti-CD28 (0.5 μg mL−1, clone 37.51, Biolegend, 102116) antibodies40. For each T cell differentiation,

(7) Targeting ubiquitin-specific protease 8 sensitizes anti-programmed death-ligand 1 immunotherapy of pancreatic cancerCell death and differentiationFebruary 1, 2023Yang H, Zhang X, Lao M, Sun K, He L, Xu J, Duan Y, Chen Y, Ying H, Li M, et al.used to isolate CD8 + T cells from mouse spleens, which were activated with 3.5 μg/ml CD3 antibody (100359, BioLegend, San Diego, CA, USA) and 1 μg/ml CD28 antibody (102116, BioLegend) for 48 h. KPC cells were

(8) High glucose promotes regulatory T cell differentiationPloS oneJanuary 1, 2023Pitmon E, Meehan EV, Ahmadi E, Adler AJ, Wang K.then cultured in 96-well round bottom plates with 0.5 μg/mL plate-bound anti-CD3 (Biolegend, Cat # 100340). T cells were cultured in RPMI medium (ThermoFisher Scientific, Cat #11879020) supplemented with indicated

(9) Protocol to isolate and enrich mouse splenic naive CD4+ T cells for in vitro CD4+CD8αα+ cell inductionSTAR ProtocolsDecember 1, 2022Yosuke Harada, Kentaro Miyamoto, Tomohisa Sujino1:200)",BD Biosciences,Cat# 553142; RRID: AB_394657 CD3ε (clone: 145-2C11) (dilution 1:3000),BioLegend,Cat# 100359; RRID: AB_2616673 CD28 (clone: 37.51) (dilution 1:1000),BioLegend,Cat# 102116; RRID: AB_11147170 ,, "Chemicals,

(10) Annexin A2/TLR2/MYD88 pathway induces arginase 1 expression in tumor-associated neutrophilsJ Clin InvestNovember 15, 2022Huajia Zhang, Xiaodong Zhu, Travis J. Friesen, Jeff W. Kwak, Tatyana Pisarenko, Surapat Mekvanich, Mark A. Velasco, Timothy W. Randolph, Julia Kargl, A. McGarry HoughtonPurified anti–mouse CD3 and purified anti–mouse CD28 antibodies were purchased from BioLegend (catalog numbers 100339 and 102122, respectively). IFN-γ, ANXA2, and ARG1 ELISAs were performed using R&D Systems Quantikine

(11) LRRC15+ myofibroblasts dictate the stromal setpoint to suppress tumour immunityNatureSeptember 28, 2022Akshay T. Krishnamurty, Justin A. Shyer, Minh Thai, Vineela Gandham, Matthew B. Buechler, Yeqing Angela Yang, Rachana N. Pradhan, Amber W. Wang, Patricia L. Sanchez, Yan Qu, et al.96-well flat-bottomed plates were coated overnight at 4 °C with 10 µg ml–1 of anti-CD3 (BioLegend, 100340, clone 145-2C11) and washed once with PBS. Relevant primary CAFs were sorted from digested KPR subcutaneous

(12) Succinate uptake by T cells suppresses their effector function via inhibition of mitochondrial glucose oxidationCell reportsAugust 16, 2022Gudgeon N, Munford H, Bishop EL, Hill J, Fulton-Ward T, Bending D, Roberts J, Tennant DA, Dimeloe S.anti-CD28 monoclonal antibodies (plate-bound, 1 and 5 μg/mL, Biolegend, anti-CD3 Clone 145-2C11 Cat# 100340; anti-CD28 Clone 37.51 Cat# 102116). Flow cytometry analysis of protein expression, degranulation and

(13) Antitumor efficacy of (90)Y-NM600 targeted radionuclide therapy and PD-1 blockade is limited by regulatory T cells in murine prostate tumorsJournal for immunotherapy of cancerAugust 1, 2022Potluri HK, Ferreira CA, Grudzinski J, Massey C, Aluicio-Sarduy E, Engle JW, Kwon O, Marsh IR, Bednarz BP, Hernandez R, et al.incubated in 0.1 M sodium phosphate buffer with 40 µg of total protein comprised of 10% anti-CD3 (Biolegend 100339), 10% anti-CD28 (Biolegend 102115), and 80% IgG for CD3/28/IgG beads, or 10% PD-L1 and 70% IgG for CD3/28/PD-L1

(14) AMPK suppresses Th2 cell responses by repressing mTORC2Experimental & molecular medicineAugust 1, 2022Pandit M, Timilshina M, Gu Y, Acharya S, Chung Y, Seo SU, Chang JH.penicillin–streptomycin. Naive CD4+ T cells were activated with plate-bound anti-CD3 (5 μg/mL; Cat #100359; BioLegend, CA, USA) and soluble anti-CD28 (1 μg/mL; Cat # BE0015-1; Bio X Cell, NH, USA). For in vitro

(15) The antimicrobial peptide cathelicidin drives development of experimental autoimmune encephalomyelitis in mice by affecting Th17 differentiationPLoS biologyAugust 1, 2022Smith KJ, Minns D, McHugh BJ, Holloway RK, O'Connor R, Williams A, Melrose L, McPherson R, Miron VE, Davidson DJ, et al.cells were differentiated for 48 hours in the presence of plate-bound αCD3 (2.5 μg/ml; Biolegend, #100339), rIL-6 (20 ng/ml; Biolegend, #575706), rIL-23 (20 ng/ml; Biolegend, #589006), and rTGFβ (3 ng/ml; Biolegend,

(16) Low-dose interleukin-2 reverses chronic migraine-related sensitizations through peripheral interleukin-10 and transforming growth factor beta-1 signalingNeurobiology of PainAugust 1, 2022Zhaohua Guo, Jintao Zhang, Xuemei Liu, Jacqueline Unsinger, Richard S Hotchkiss, Yu-Qing Caocells per well) and stimulated overnight at 37 °C with antibodies against CD3 (1 µg/ml, Biolegend, 100340, clone 145-2C11) and CD28 (5 µg/ml, Biolegend, 102116, clone 37.51) in 1 ml complete RPMI1640 media

(17) Androgen conspires with the CD8 ⁺ T cell exhaustion program and contributes to sex bias in cancerScience ImmunologyJuly 1, 2022Hyunwoo Kwon et al.manufacturer’s recommendations. Splenocytes were then stimulated using plate-bound 5 μg/mL CD3 (BioLegend, 100359) and 2 μg/mL CD28 (BioLegend, 102121) plus 40 ng/mL IL2 (NIH) for 72 hours. Afterwards, cells were washed

(18) Naïve Primary Mouse CD8+ T Cells Retain In Vivo Immune Responsiveness After Electroporation-Based CRISPR/Cas9 Genetic EngineeringFrontiers in ImmunologyJune 30, 2022Petra Pfenninger, Laura Yerly, Jun Abeactivated CD8+ T cells, T-25 or T-75 flasks were coated with 5 µg/mL anti-CD3ϵ antibodies (clone 145-2C11; 100340, BioLegend) in phosphate-buffered saline (PBS) for 16–24 hr at 4°C before seeding CD8+ T cells. Immediately

(19) An innate IL-25-ILC2-MDSC axis creates a cancer-permissive microenvironment for Apc mutation-driven intestinal tumorigenesisScience immunologyJune 3, 2022Jou E, Rodriguez-Rodriguez N, Ferreira AF, Jolin HE, Clark PA, Sawmynaden K, Ko M, Murphy JE, Mannion J, Ward C, et al.added to the ILC2-SNT-M-MDSC culture, and stimulated for 3 days with plate-coated anti-CD3ε (Biolegend, 100360) and 2 μg/mL soluble anti-CD28 (Bio X Cell, BE0015-1) at 37°C. Cell Stimulation Cocktail (eBioscience,

(20) Immune-mediated neurodegenerative trait provoked by multimodal derepression of long-interspersed nuclear element-1iScienceMay 20, 2022Takahashi F, Zhang C, Hohjoh H, Raveney B, Yamamura T, Hayashi N, Oki S.Pyruvate,Gibco,Cat#11360-070 2-ME,Gibco,Cat#21985-023 Glutamax,Gibco,Cat#35050-061 anti-CD3(2C11),Biolegend,Cat#100340; RRID:AB_11149115 CellTiter-Glo 2.0 reagent,Promega,Cat#G924B ,, Experimental models: Organisms/strains,Experimental models

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eLife

Nature communications

Neurobiology of Pain

Nature communications

Journal for immunotherapy of cancer

Ultra-LEAF(TM) Purified anti-mouse CD3epsilon from BioLegend

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Ultra-LEAF(TM) Purified anti-mouse CD3epsilon from BioLegend

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