Anti mouse cd3e

Manufactured by BD

Sourced in United States

Anti-mouse CD3e is a monoclonal antibody that binds to the CD3 epsilon subunit of the T cell receptor complex in mice. It is used for the detection and isolation of mouse T cells in research applications.

Automatically generated - may contain errors

Lab products found in correlation

15 protocols using anti mouse cd3e

Isolation and Activation of CD8+ T Cells

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

Spleens isolated from P14, Pmel, or OT-1 TCR transgenic mice were dissociated in complete RPMI media [RPMI 1640 (Gibco) + 10% fetal bovine serum (FBS; Gibco) + 1% penicillin-streptomycin (Gibco)], and red blood cells were lysed using RBC lysis buffer (BioLegend). CD8⁺ T cells were isolated using a CD8a⁺ T cell isolation kit (Miltenyi Biotec). For T cell activation, isolated CD8 T cells were cultured in T cell media [complete RPMI media supplemented with 1× nonessential amino acids (Gibco) + 1 × 10⁻³ M sodium pyruvate (Gibco) + 0.05 × 10⁻³ M 2-mercaptoethanol (Sigma-Aldrich)] supplemented with soluble anti-mouse CD28 (5 μg/ml; BD Pharmingen) and rhIL-2 (30 U/ml; Roche) at 1 × 10⁶ cells/ml in wells coated with anti-mouse CD3e (3 μg/ml; BD Pharmingen).

Su F.Y., Zhao Q.H., Dahotre S.N., Gamboa L., Bawage S.S., Silva Trenkle A.D., Zamat A., Phuengkham H., Ahmed R., Santangelo P.J, & Kwong G.A. (2022). In vivo mRNA delivery to virus-specific T cells by light-induced ligand exchange of MHC class I antigen-presenting nanoparticles. Science Advances, 8(8), eabm7950.

+ Open protocol

+ Expand

Quantifying Cytotoxic T Cell Responses Against Tumor Antigens

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

EL4 and EG7-OVA cells (ATCC) were grown in RPMI 1640 supplemented with 10% FBS and 25mM HEPES (Gibco). EG7-OVA cultures were supplemented with G418 (0.4 mg/ml, InvitroGen). CD8 T cells were isolated from OT1 (Jackson Labs) splenocytes by MACS using CD8a Microbeads (Miltenyi). Cells were activated by seeding in 96-well plates pre-coated with anti-mouse CD3e (1 μg/ml working concentration, Clone: 145–2C11, BD) and anti-mouse CD28 (2 μg/ml working concentration, Clone: 37.51, BD) at 2×10⁶ cells/ml in RPMI 1640 supplemented with 10% FBS, 100U/ml penicillin-streptomycin, 1X non-essential amino acids (Gibco), 1mM sodium pyruvate, 0.05mM 2-mercaptoethanol, and 30U/ml hIL-2 (Roche). After 2 days, cells were washed and transferred to uncoated plates. On day 5, 1 × 10⁶ activated OT1 T cells were coincubated with 1×10⁶ EL4 or EG7-OVA cells for 2 hours at 37 °C and stained for GzmB using anti-mouse GzmB (Clone: NGZB, eBioScience) and Intracellular Fixation & Permeabilization Buffer Set (eBioScience, 88–8824-00). To measure GzmB activity inside target cells, we coincubated activated OT1 CD8 T cells with EL4 and EG7-OVA target cells at various T cell to target cell ratios and stained using GranToxiLux Kit (OncoImmunin, GTL702–8). To measure secretory GzmB, we collected coculture supernatant of OT1 with target cells and performed ELISA with Granzyme B Mouse ELISA Kit (eBioScience, BMS6029).

Mac Q.D., Mathews D.V., Kahla J.A., Stoffers C.M., Delmas O.M., Holt B.A., Adams A.B, & Kwong G.A. (2019). Non-invasive early detection of acute transplant rejection via nanosensors of granzyme-B activity. Nature biomedical engineering, 3(4), 281-291.

+ Open protocol

+ Expand

Isolation and Activation of CD8+ T Cells

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

Spleens from P14, pmel, or OT1 TCR transgenic mice (Jackson Labs) were dissociated in R10 media (RPMI 1640 (Gibco) + 10% FBS (Gibco) + 1% pen/strep (Gibco)), and red blood cells were lysed using RBC Lysis Buffer (Biolegend). CD8+ T cells were isolated using a CD8a+ T Cell Isolation Kit (Miltenyi Biotec). For pMHC liposome activation in vitro, T cells were cultured at 2×10⁶ cells/ml in T cell media (R10 supplemented with 1X non-essential amino acids (Gibco) + 1 mM sodium pyruvate (Gibco) + 0.05 mM 2-mercaptoethanol (Sigma)), and liposomes were added at 1 μg/ml. For antibody-activated controls, T cells were cultured in T cell media supplemented with soluble anti-mouse CD28 (2 μg/ml, Clone: 37.51, BD Pharmingen) and 30 U/ml rhIL-2 (Roche) at 2×10⁶ cells/ml in wells coated with anti-mouse CD3e (1 μg/ml, Clone: 145–2C11, BD Pharmingen).

Dahotre S.N., Romanov A.M., Su F.Y, & Kwong G.A. (2021). Synthetic Antigen-Presenting Cells for Adoptive T Cell Therapy. Advanced therapeutics, 4(8), 2100034.

+ Open protocol

+ Expand

TCR and Cytokine Activation Assays

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

For TCR or cytokine activation, splenocytes were cultured in antibody-coated plates (anti-mouse CD3e; BD PharMingen, 5 μg/ml) or in complete RPMI medium containing IL-2 (200 U/ml) or IL-15 (100 ng/ml) for 48 h. Cells were harvested, stained for surface markers and analyzed by flow cytometry. For incubation with tumor cells, BALB/c WT or DGKζ^−/− splenocytes were co-cultured with A20 cells (1:1) for 14 h, stained for surface markers and analyzed by flow cytometry.
For degranulation assays, IL-2-differentiated cells from BALB/c WT or DGKζ^−/− mice were incubated with A20 cells (1:1 proportion) during 4 h in presence of anti-CD107a-PE (BD Pharmigen). Then cells were washed, stained for surface markers and analyzed by flow cytometry.

Andrada E., Liébana R, & Merida I. (2017). Diacylglycerol Kinase ζ Limits Cytokine-dependent Expansion of CD8+ T Cells with Broad Antitumor Capacity. EBioMedicine, 19, 39-48.

+ Open protocol

+ Expand

Cytotoxicity Assay of Activated T Cells

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

B16-F10 cells (ATCC) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (Thermo). CD8+ T cells were isolated from either OT1 or Pmel (Jackson Labs) splenocytes by MACS using CD8a Microbeads (Miltenyi). Cells were activated by seeding in 96-well plates pre-coated with anti-mouse CD3e (1 μg/ml working concentration, Clone: 145-2C11, BD) and anti-mouse CD28 (2 μg/ml working concentration, Clone: 37.51, BD) at 2×10⁶ cells/ml in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin-streptomycin, 1X non-essential amino acids (Gibco), 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, and 30 U/ml hIL-2 (Roche). After 2 days, cells were washed and transferred to untreated culture flasks for expansion. Between day 4 to 6 after activation, activated T cells were washed before being coincubated with 3×10⁴ B16 target cells at various T cell to effector cell ratios. After 48 hours, coculture supernatants were collected for LDH and GzmB measurements by the Pierce LDH Cytotoxicity Assay Kit (Thermo) and GzmB Mouse ELISA Kit (Thermo, BMS6029) respectively. To assess sensor activation during T cell killing, cocultured of T cells and target cells were spiked in with either αPD1-GS, αPD1 conjugated with control peptide (LQRIYK), and unconjugated αPD1. After 48 hours, fluorescence of coculture supernatant were measured using Cytation 5 plate reader (Biotek).

Mac Q.D., Sivakumar A., Phuengkham H., Xu C., Bowen J.R., Su F.Y., Stentz S.Z., Sim H., Harris A.M., Li T.T., Qiu P, & Kwong G.A. (2022). Urinary detection of early responses to checkpoint blockade and of resistance to it via protease-cleaved antibody-conjugated sensors. Nature biomedical engineering, 6(3), 310-324.

+ Open protocol

+ Expand

T Cell Activation Assay Protocol

Cited 1 time

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

Ninety-six well culture plates (Sigma-Aldrich, St. Louis, USA) were coated with anti-mouse-CD3e (clone: 145-2C11, BD Biosciences) at 10 μg/ml and stored overnight at 4°C. As described in previous studies in more detail, after isolation, 2 × 10⁵ CD4⁺CD62L^high and CD4⁺CD62L_low cells were coincubated with either 2 × 10⁵ DX5⁺NKT cells or CD8⁺ T cells in 200 μl RPMI culture medium (Gibco, Paisley, UK) in either coated or uncoated wells [7 (link)]. For further stimulation, 5 μg/ml anti-mouse-CD28 (clone: 37.51, BD Biosciences) and 2000 IU/ml IL-2 (PeproTech, Rocky Hill, USA) were added [31 (link)]. Control cultures of CD4⁺CD62L^high, CD4⁺CD62L_low, DX5⁺NKT cells, and CD8⁺T cells only were incubated at 4 × 10⁵ cells in 200 μl RPMI under the same conditions.

Werner J.M., Damian M., Farkas S.A., Schlitt H.J., Geissler E.K, & Hornung M. (2018). Murine DX5+NKT Cells Display Their Cytotoxic and Proapoptotic Potentials against Colitis-Inducing CD4+CD62Lhigh T Cells through Fas Ligand. Journal of Immunology Research, 2018, 8175810.

+ Open protocol

+ Expand

Murine Tumor Immune Profiling

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

For murine samples, mice with tumors were killed according to the institutional ethical guidelines and femurs, tibias, and tumors were collected. Signal cell of suspension of tumor was obtained by using the Tumor Dissociation Kit (Miltenyi Biotech, Bergish Gladbach, Germany, 130-096-730) according to the manufacturer’s instructions. Antibodies (BD Pharmingen, San Diego, CA, USA) used are listed: anti-mouse-CD3e (BD Pharmingen, 566494), anti-mouse-CD45 (BD Pharmingen, 553079), anti-mouse-CD8e (BD Pharmingen, 553035), anti-mouse-CD11b (BD Pharmingen, 550993), anti-mouse-Ly-6G/Ly-6C (BD Pharmingen, 553129), Anti-mouse-F4/80 (BD Pharmingen, 565410). For cell apoptosis assays, apoptosis cells were measured by a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s protocol. Data were acquired using FACS Calibur (BD Biosciences), and analyzed using Cell Quest Pro software (BD Biosciences)^{46 (link)}.

Xia S., Wu J., Zhou W., Zhang M., Zhao K., Liu J., Tian D, & Liao J. (2021). SLC7A2 deficiency promotes hepatocellular carcinoma progression by enhancing recruitment of myeloid-derived suppressors cells. Cell Death & Disease, 12(6), 570.

+ Open protocol

+ Expand

Colonic NKT Cell Isolation and Characterization

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

Single cell suspensions were prepared from the colon tissues and incubated in DMEM (Dulbecco’s modified Eagle’s medium) with collagenase 1 (Sigma-Aldrich) and dispase (Sigma-Aldrich) for 1 hour. After incubation, the cells were collected by filtering the cell solution through a 40 μm strainer. The flow cytometry analysis was conducted on 5×10⁴ cells to detect each marker. Cells were incubated for 20 minutes in the presence of an appropriate dose of antibodies in fluorescence-activating cell sorting (FACS) buffer. In this study, we used anti-mouse CD3e (BD bioscience), NK1.1 (BD bioscience), and Cd1d tetramer (Proimmune Ltd., Oxford, UK) to measure the population of NKT cells. Colonic NKT cells were sorted by first enriching the stained cells for CD3+ leukocytes and the CD3-enriched samples were then sorted with anti-NK1.1 and Cd1d tetramer. Cell sorting was conducted using a FACS Aria 1 instrument (BD Bioscience). The percentages of cell populations were calculated by acquiring a total of 10,000 events.

Lee C., Hong S.N, & Kim Y.H. (2020). A glycolipid adjuvant, 7DW8-5, provides a protective effect against colonic inflammation in mice by the recruitment of CD1d-restricted natural killer T cells. Intestinal Research, 18(4), 402-411.

+ Open protocol

+ Expand

Quantifying Cytotoxic T Cell Responses Against Tumor Antigens

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

+ Open protocol

+ Expand

Isolation and Characterization of Regulatory T Cells

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

The CD4⁺CD25⁺ Regulatory T cell Isolation Kit and CD8⁺ T cell Isolation Kit were purchased from Miltenyi Biotec (Auburn, CA). Anti-mouse CD45R/B220 antibody used for preparation of bone marrow-derived dendritic cells was purchased from BD Bioscience (San Diego, CA), while antibodies directed against CD4 (GK1.5), CD8 (Lyt-2), and HB-32 were a kind gift from Dr. Xu of the University of Alabama at Birmingham. Dynabeads coupled with anti-rat IgG antibodies were purchased from Invitrogen (Carlsbad, CA). IL-2, IL-4, DNFB and lipopolysaccharide (LPS) were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mouse CD3e, anti-mouse CD28 and GM-CSF were purchased from BD Bioscience (San Diego, CA). Mouse-specific ELISA kits for TGF-β, IL-10, and IFNγ were purchased from eBioscience (San Diego, CA). The GSPs were obtained from the Kikkoman Corporation (Japan) and the chemical composition of this product has been described previously [25 (link), 26 (link)]. Experimental diet containing GSPs (0.5%, w/w) was prepared commercially in pellet form in the AIN76A-powdered control diet by TestDiet (Richmond, IN) using the GSPs that we provided.

Vaid M., Prasad R., Singh T, & Katiyar S.K. (2017). Dietary grape seed proanthocyanidins inactivate regulatory T cells by promoting NER-dependent DNA repair in dendritic cells in UVB-exposed skin. Oncotarget, 8(30), 49625-49636.

+ 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!