Thy1.1+-enriched cells after lentiviral gRNA vector transduction were activated in vitro with 3 µg/ml plate-bound α-CD3ε (145-2C11, Biolegend) and 1 µg/ml of soluble α-CD28 (37.51, Biolegend). Cells were collected after 3–7 d after activation for preparation of whole cell lysates in radioimmunoassay precipitation buffer for subsequent immunoblot analysis to assess the efficiency of gene inactivation. Total cell lysates equivalent to 2–5 × 105 cells were loaded into each lane for SDS-PAGE and subsequent protein transfer to a polyvinylidene difluoride membrane. Antibodies for Western blotting against HDAC3 (2632, 85057), Blimp-1 (9115), Runx3 (9647), and GAPDH (2118) were purchased from Cell Signaling Technology. Membranes were incubated with primary antibodies overnight at 4°C with gentle shaking at a dilution of 1:1,000 in SuperBlock T20 (TBS) (Thermo Fisher Scientific), except for α-GAPDH antibodies, which were diluted 1:5,000. Membranes were then incubated with secondary goat α-rabbit HRP-conjugated antibodies (1:10,000; Cell Signaling Technology) for 1 h at room temperature and then treated with enhanced chemiluminescence reagent (Thermo Fisher Scientific). Protein bands were visualized on autoradiography film (Denville Scientific), and protein KO levels were quantified from scanned film images using FIJI software (National Institutes of Health).
α cd3ε 145 2c11
Manufactured by BioLegend
Sourced in United States
The α-CD3ε (145-2C11) is a monoclonal antibody that binds to the CD3 epsilon chain of the T cell receptor complex. It is commonly used in cell culture and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Soluble αcd28 37.51, by BioLegend (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Gapdh 2118, by Cell Signaling Technology (1 mentions)
Superblock t20 tbs, by Thermo Fisher Scientific (1 mentions)
α cd11b fitc, by BioLegend (1 mentions)
Anti il 2, by R&D Systems (1 mentions)
Anti ly6g pe, by BD (1 mentions)
α cd4 apc, by BioLegend (1 mentions)
Anti ly6c percp, by Thermo Fisher Scientific (1 mentions)
αcd4 pe, by BioLegend (1 mentions)
α cd25 pe, by BD (1 mentions)
αcd11c apc, by BioLegend (1 mentions)
Mouse igg2b, by BioLegend (1 mentions)
Pyruvate, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Mem neaa, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Torin1, by Selleck Chemicals (1 mentions)
Mouse na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
4 protocols using α cd3ε 145 2c11
1
Assessing Gene Knockout Efficiency
2
Multiparameter Flow Cytometry Protocol
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Anti-CD11b-PacBlu, αCD11b-FITC, αCD4-PE, αCD4-APC, αCD11c-APC, αCD11c-PE, αTCRvα2-PE, and αCD3ε(145-2C11) were purchased from BioLegend (San Diego, CA, USA). Anti-Ly6C-PerCP and αFoxP3-APC were products of eBioscience (San Jose, CA, USA). Anti-Ly6G-PE, αNK1.1-PE, αCD8α-PacBlu, αCD25-PE, and αCD103-Alexa Fluor 647 were products from BD Biosciences (San Jose, CA, USA). Flt3L-Fc fusion protein was purchased from BioXCell (West Lebanon, NH, USA). Anti-IL-2 was purchased from R&D Systems (Minneapolis, MN, USA). Fc-GITR-L fusion protein was produced as described previously (9 (link)).
3
Antibody-Mediated T Cell Activation
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20μg of purified αCD3ε (145–2C11) (Biolegend, San Diego, CA) or Mouse IgG2b (Biolegend, San Diego, CA) were intraperitoneally injected. Three or five days later the mice were euthanized. Lamina propria cells were isolated and analyzed by flow cytometry [20 (link)].
4
Naïve CD4+ T Cell Differentiation
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Naïve CD4+ T cells were isolated by negative selection with mouse Naïve CD4+ T cell isolation kit (Miltenyi). 1 × 105 cells were cultured for 4 days in 96-well plates precoated with 1 μg/ml of α-CD3ε (145-2C11; Biolegend) and 1 μg/ml of soluble α-CD28 (37.51; Biolegend) in RPMI 1640 media supplemented with 10% FBS, 1% penicillin/streptomycin, 1% pyruvate (11360070; Thermo Fisher Scientific), 1% MEM/NEAA (11140050; Thermo Fisher Scientific), 2.5% HEPES (15630080; Thermo Fisher Scientific), and 55 μM β-mercaptoethanol (21985023; Thermo Fisher Scientific) under CD4+CD8αα+ T cell conditions: RA (10 nM, 0064-1G, Tokyo Chemical Industry), TGFβ (2 ng/ml, 7666-MB-005, R&D systems), IFNγ (20 ng/ml, 315-05, Peprotech). For the treatment with inhibitors, Rapamycin (1 μM, Sigma-Aldrich), Torin-1 (1 μM, Selleckchem) and 2DG (1 mM, Sigma-Aldrich) were added into the cell culture. In some experiments, cells were placed inside a sealed air tight container which contains an anaeropack (Mitsubishi Gas Company, Tokyo, Japan). The anaeropack contains a gas-controlling reagent which absorbs oxygen and generates carbon dioxide resulting in a hypoxic atmosphere (8% O2).
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