Anti cd3 mab clone 145 2c11

Manufactured by BioLegend

Sourced in United States

Anti-CD3 mAb (clone 145-2C11) is a monoclonal antibody that binds to the CD3 complex on T cells. It is commonly used in research applications to stimulate and activate T cells.

Automatically generated - may contain errors

Lab products found in correlation

5 protocols using anti cd3 mab clone 145 2c11

In vitro T cell activation assay

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

Single cells suspension from the spleens of Listeria infected mice were stimulated in vitro in 24-well culture plates with anti-CD3 MAb, clone 145-2C11 from Biolegend (1 μg/mL) and anti-CD28 MAb, clone 37.51 from Biolegend (0.5 μg/mL) plus IL-2 (50 U/mL) or with IL-2 only. Lung or spleen single cell suspensions from M. tuberculosis-infected mice were stimulated with TB10.4_4-11 and 32A_309-318 peptides (10 uM) in solution plus IL-2 (50 U/mL). In some experiments, the combination of soluble anti-CD3/CD28 MAbs was used instead of peptides. Media was replenished with complete RPMI cRPMI; 10% heat-inactivated FCS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine, 50 mg/mL streptomycin, and 50 U/mL penicillin (all from Invitrogen), 0.05 mM 2-mercaptoethanol (Gibco) and IL-2 (50 U/mL) every third day. Cells were analyzed by flow cytometry after 7 days.

Thakur P., Sutiwisesak R., Lu Y.J, & Behar S.M. (2022). Use of the Human Granulysin Transgenic Mice To Evaluate the Role of Granulysin Expression by CD8 T Cells in Immunity To Mycobacterium tuberculosis. mBio, 13(6), e03020-22.

+ Open protocol

+ Expand

CIA Mice Splenocyte Proliferation Assay

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

On day 28 after the second immunization, mononuclear splenocytes (1 × 10⁵/well) of CIA mice were seeded onto 96-well tissue culture plates (Corning, Corning, NY, USA) in 10% FBS/RPMI 1640 medium and stimulated with CII (30 μg/ml) or anti-CD3 mAb (clone 145-2C11, 1 μg/ml; BioLegend) in the presence of antimouse OX40L mAb (clone RM134L; BioLegend). Rat immunoglobulin G (IgG) (clone RTK4530; BioLegend) was added as a control. After 72 h of incubation, cell proliferation was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Mashikimachi, Japan). Supernatant cytokine levels were assessed using a mouse Th1/Th2/Th17 cytokine kit (BD Biosciences) according to the manufacturer’s instructions.

Jiang J., Liu C., Liu M., Shen Y., Hu X., Wang Q., Wu J., Wu M., Fang Q, & Zhang X. (2017). OX40 signaling is involved in the autoactivation of CD4+CD28− T cells and contributes to the pathogenesis of autoimmune arthritis. Arthritis Research & Therapy, 19, 67.

+ Open protocol

+ Expand

Regulatory T Cell Suppression Assay

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

Single cell suspensions from spleen and LNs of CnB ΔTreg^het, CD28 ΔTreg^het or Foxp3^YFP-Cre/wt mice were enriched for Treg through immunomagnetic selection of CD4⁺ CD25⁺ cells (Miltenyi). YFP⁺ CD44^low CD62L^hi cTreg and YFP⁺ CD44^hi CD62L^neg eTreg were further purified by FACS. Varying numbers of cTreg or eTreg were added to 2.5×10⁴ T-cell depleted splenocytes as APC and 1×10⁴ CD4⁺ CD25^– T cells labeled with 5µM CellTrace Violet (CTV) as responder cells, and stimulated with 250 ng/ml of anti-CD3 mAb (clone 145-2c11, Biolegend). Responder cell proliferation (CTV dilution) and absolute numbers of remaining Treg was read out after 72h. To quantify proliferation, we extrapolated the number of progenitor cells giving rise to each CTV peak by dividing the number of cells in each peak by the number of cells that would have originated from a single precursor (e.g. 4 cells in the peak corresponding to two divisions would have derived from 1 progenitor). To compute the T cell fold increase, we then divided the total number of recorded Treg by the extrapolated total number of progenitors. Percentage of suppression was scaled from 0 (proliferation of responders in absence of Treg) to 100 (complete absence of proliferation).
To follow cTreg proliferation, an identical assay was set up, but Treg, not responder T cells, were labeled with CTV.

Marangoni F., Zhang R., Mani V., Thelen M., Ali Akbar N.J., Warner R.D., Äijö T., Zappulli V., Martinez G.J., Turka L.A, & Mempel T.R. (2018). Tumor tolerance-promoting function of regulatory T cells is optimized by CD28, but strictly dependent on calcineurin. Journal of immunology (Baltimore, Md. : 1950), 200(10), 3647-3661.

+ Open protocol

+ Expand

T Cell-Microglia Interaction Assay

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

Spleens of C57BL/6J mice were gently dissociated into single-cell suspensions, and red blood cells were removed using Ammonium-Chloride-Potassium (ACK) lysis buffer (Gibco). Splenocytes were labeled with 5(6)-Carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE, Molecular probes – Invitrogen, 5 μmol l⁻¹) at 37 °C followed by staining with anti-CD3 mAb (clone 145-2C11, Biolegend, 2 μg ml⁻¹) for 30 min on ice. Responder CFSE⁺ CD3⁺ T cells were sorted using FACS Aria II (BD Biosciences) with a purity > 98%. T cells were stimulated with mouse T cell-activator CD3 and CD28 magnetic beads (Dynabeads-Gibco). Stimulated CFSE⁺ CD3⁺ T cells were co-cultured in round bottom 96-well plates with FCRLS⁺ spinal cord microglia for 72 h at 37 °C in a 5% CO2 incubator. T cell-to-microglia ratio was 4:1 in a final volume of 200 μl per well in DMEM media supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin-Streptomycin (PS, 10,000 U ml⁻¹), L-Glutamine 2 mmol l⁻¹, sodium pyruvate 1 mmol l⁻¹, non-essential amino acids 0.1 mmol l⁻¹, HEPES 5 mmol l⁻¹ and 2-Mercaptoethanol 0.05 mmol l⁻¹. T cell proliferation was measured by flow cytometric analysis by CFSE dilution on CD3⁺ T cells. Soluble recombinant ApoE (rApoe) was added into the media at 100 ng ml⁻¹.

Krasemann S., Madore C., Cialic R., Baufeld C., Calcagno N., El Fatimy R., Beckers L., O’Loughlin E., Xu Y., Fanek Z., Greco D.J., Smith S.T., Tweet G., Humulock Z., Zrzavy T., Conde-Sanroman P., Gacias M., Weng Z., Chen H., Tjon E., Mazaheri F., Hartmann K., Madi A., Ulrich J., Glatzel M., Worthmann A., Heeren J., Budnik B., Lemere C., Ikezu T., Heppner F.L., Litvak V., Holtzman D.M., Lassmann H., Weiner H.L., Ochando J., Haass C, & Butovsky O. (2017). The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity, 47(3), 566-581.e9.

+ Open protocol

+ Expand

T Cell Proliferation Assay

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

After 4 days of stimulation of CD25-depleted LN cells with D665 beads CD4 + T cells were purified and the beads depleted by magnetic separation (Miltenyi) as described in the previous section. The CD4 + T cells were then cultured for 3 days at 1 × 10 6 /mL in the presence of 10 -7 M recombinant human IL-2 (Proleukin; Novartis). The precultured cells or freshly isolated CD4 + CD25 - T cells for comparison were then seeded at a density of 2 × 10 4 cells/well of a 96-well round bottom plate (Greiner) together with 5 × 10 4 Dynabeads Pan Mouse IgG (Invitrogen) coated either with 10 μg/mL normal mouse Ig (Sigma) or 10 μg/mL anti-CD3 mAb (clone 145-2C11; Biolegend) and 1 μg/mL anti-CD28 mAb (clone E18 [45] ) according to the manufacturer's instructions. Where indicated Proleukin, recombinant mouse IL-7 (R&D Systems) and recombinant mouse IL-15 (Biolegend) were added at a final concentration of 10 -6 M each. The total volume per well was 50 μL. All cultures were set up in triplicates. 3 H-thymidine was added for the last 16 h of the 3-day culturing period.

Beyersdorf N., Werner S., Wolf N., Hünig T, & Kerkau T. (2015). In vitro polyclonal activation of conventional T cells with a CD28 superagonist protects mice from acute graft versus host disease. European journal of immunology, 45(7).

+ 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!