All antibodies were purchased from commercial sources: CD3-phycoerythrin (PE), CD4-allophycocyanin (APC), CD4-peridinin-chlorophyll–protein complex (PerCP), CD19-PE, CD3-APC, CD3-fluorescein isothiocyanate (FITC), CD8-PE, NK1.1-APC, F4/80-PE, CD11c-PE, CD11c-APC, IL-4-PE, IFN-γ-APC, TNFα-FITC, CD69-APC, anti-CD3 Abs, and anti-CD28 Abs from eBioscience; Rabbit polyclonal GIT2 Abs, anti-phospho-PAK1/2 (Ser199/204 of PAK1 and Ser192/197 of PAK2 Abs, and anti-phospho-c-Cbl (Y774) Abs from Cell Signaling; Anti-phospho-Erk1/2 (E4) Abs, anti-PAKα, anti-Erk1/2 (K23) from Santa Cruz.
Anti cd3 ab
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-CD3 Ab is a laboratory instrument used for the detection and analysis of CD3, a protein complex found on the surface of T cells. It functions as a tool for researchers to study T cell biology and immune system responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd28 ab, by Thermo Fisher Scientific (4 mentions)
Rmil 2, by Thermo Fisher Scientific (2 mentions)
Miltenyi beads, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Cd3 phycoerythrin, by Thermo Fisher Scientific (1 mentions)
Cd4 allophycocyanin, by Thermo Fisher Scientific (1 mentions)
Cd3 fluorescein isothiocyanate, by Thermo Fisher Scientific (1 mentions)
Cd3 apc, by Thermo Fisher Scientific (1 mentions)
Cd8 pe, by Thermo Fisher Scientific (1 mentions)
Cd19 pe, by Thermo Fisher Scientific (1 mentions)
Ifn γ apc, by Thermo Fisher Scientific (1 mentions)
Tnfα fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by Thermo Fisher Scientific (1 mentions)
Nk1.1 apc, by Thermo Fisher Scientific (1 mentions)
Cd11c apc, by Thermo Fisher Scientific (1 mentions)
Cd69 apc, by Thermo Fisher Scientific (1 mentions)
10 protocols using anti cd3 ab
1
Multiparametric Immune Cell Analysis
2
CFSE-Based T Cell Proliferation Assay
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CellTrace 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR, USA) labeled human T cells were plated at 2 × 105 cells/well in 1 μg/mL of anti-CD3 Abs (eBioscience, San Diego, CA, USA)-coated plates in the presence of 0.5 μg/mL of anti-CD28 Abs (eBioscience) with or without indicated concentrations of recombinant SRA-ECD protein. In some case, 100 U/ml of IL-2 was added in the incubation. Proliferation was analyzed using FACS based on the dilution of fluorescence intensity. Supernatants were collected at 48 hours after the stimulation for cytokine assays using commercial ELISA kit (eBioscience).
3
Regulatory T Cell Isolation and Activation
4
Isolation and Stimulation of Memory T Cells
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All protocols were approved by the Yale Institutional Review Board (Protocol No. 0601000969). PBMCs were isolated from leukopacks obtained from the Red Cross (Philadelphia, PA) using density centrifugation as described previously and cryopreserved in liquid nitrogen. CD4+CD45RO+ T cells were isolated from thawed cryovials using magnetic bead separation kits (Miltenyi, Charlestown, MA, USA) with HLA-DR Ab (clone L243, Novus No. NB100-77855) and CD45RA Ab-negative depletion (10 μL per cryovial, eBiosciences, 14-0458-82, San Diego, CA, USA). Where indicated, flat-bottom 96-well microtiter plates were coated with anti-CD3 Ab (1 μg/mL, eBiosciences, No. 16003785) in sterile PBS at 4°C overnight, and the following day, isolated CD4+CD45RO+ T cells (Tmem) were pretreated with soluble anti-CD28 Ab (1 μg/mL, eBiosciences, No. 16-0281-82) or SAG (15 μM) for 1 h prior to addition to anti-CD3 Ab-coated plates. Tmem were stimulated for 48 h prior to use in downstream applications including TCR deep sequencing, single-cell proteomics, mass cytometry, and passive transfer into humanized mice.
5
Activated CD4+ T Cell RNA Isolation
6
T Cell Proliferation Assay for Nf1 Mice
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Following the manufacturer’s recommendations, CD3+ T cells were purified from the lymph nodes of Nf1+/+ and Nf1+/− mice (age 3–6 months) using pan-T-cell isolation kit (Miltenyi Biotec, Auburn, CA). Purified CD3+ T cells (2 × 105/well) were cultured for 2 days in 96-well plates (in triplicate) precoated with indicated doses of anti-CD3 Ab (eBioscience; cat. no. 16-0031-86) alone or anti-CD28 mAb (eBioscience; cat. no. 16-0281-85). After pulsing with 3H-thymidine (1 μCi/well [0.037 MBq/well]) for 20–22 h, cells were collected and evaluated for 3H radioactivity. For each experimental group, the T cell proliferation assay was performed in two pooled biological and three technical replicates.
7
Cytokine Production by MNCs
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MNCs (5 × 105 cells) of liver or splenic origin in 200 μL of medium were cultured with either α-GalCer (100 ng/mL), ConA (1 ng/mL, Vector Laboratories, Inc., Burlingame, CA), anti-CD3 Ab (10 μg/mL, eBioscience), CpG-ODN (20 μg/mL, Hycult Biotech, Uden, The Netherlands), or LPS from E. coli 0111:B4 (10 μg/mL, Sigma-Aldrich, St. Louis, MO) in 96-well flat-bottom plates. The culture supernatants were harvested 48 hours after seeding.
8
Skin Biopsy Histology and Imaging
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Circular, 4-mm punch biopsies were excised from the nape of the neck and fixed in 4% formaldehyde and embedded in paraffin. Sections were cut at 4 μm, mounted onto slides, and stained with hematoxylin and eosin or toluidine, following standard protocols. For immunofluorescent staining, sections were deparaffinized and antigen was retrieved before staining. Anti-CD3 Ab was purchased from eBioscience and corresponding secondary Ab from Invitrogen (Thermo Fisher Scientific). Images were obtained by Zeiss Imager Z1 microscope and analyzed by Zen software (Zeiss). For quantification, 3 images were randomly selected per mouse tissue and averaged.
9
Profiling Pulmonary Immune Responses
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Mice were sacrificed for sampling BAL, lungs, spleen and blood. Total cell numbers per BAL was determined. For histopathology, lungs were fixed by inflation and immersion in paraformaldehyde (PFA; 4%) and embedded in paraffin. To evaluate airway inflammation, lung sections (4-μm thick) were stained by hematoxylin & eosin.
Pulmonary cells from air or CS-exposed mice were prepared as previously described (van der Poll and Opal, 2009 (link)) and were analyzed by flow cytometry. To analyze cytokine profiles, pulmonary cell suspensions were incubated with phorbol 12-myristate 13-acetate (PMA; 20 ng/ml) and ionomycin (500 ng/ml) for 3 h. Cells were then stained with appropriate extracellular markers, fixed, permeabilized (BD Cytofix/cytoperm, BD Bioscience), and incubated with PE-conjugated mAb against IL-22 (eBiosciences) and Alexa Fluor 647-conjugated mAb against IL-17 (Biolegend), or control rat IgG1 mAb. Cells were acquired on a Fortessa cytometer (Becton Dickinson), and analyzed using the FlowJo software.
Cytokine production was analyzed in total lung cells. For this, 5 × 105 lung cells were seeded on 96-well plates coated or not with anti-CD3 Ab (eBiosciences). Forty-eight hours later, supernatants were collected and analyzed for IFN-γ, IL-17, and IL-22 concentration by ELISA (R&D Systems).
Pulmonary cells from air or CS-exposed mice were prepared as previously described (van der Poll and Opal, 2009 (link)) and were analyzed by flow cytometry. To analyze cytokine profiles, pulmonary cell suspensions were incubated with phorbol 12-myristate 13-acetate (PMA; 20 ng/ml) and ionomycin (500 ng/ml) for 3 h. Cells were then stained with appropriate extracellular markers, fixed, permeabilized (BD Cytofix/cytoperm, BD Bioscience), and incubated with PE-conjugated mAb against IL-22 (eBiosciences) and Alexa Fluor 647-conjugated mAb against IL-17 (Biolegend), or control rat IgG1 mAb. Cells were acquired on a Fortessa cytometer (Becton Dickinson), and analyzed using the FlowJo software.
Cytokine production was analyzed in total lung cells. For this, 5 × 105 lung cells were seeded on 96-well plates coated or not with anti-CD3 Ab (eBiosciences). Forty-eight hours later, supernatants were collected and analyzed for IFN-γ, IL-17, and IL-22 concentration by ELISA (R&D Systems).
10
Regulatory T Cell Isolation and Activation
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CD4+CD25+ Treg cells were purified from spleen with Miltenyi beads, and cultured in RPMI-1640 (Gibco, Grand Island, NY) supplemented with 10% FBS (Hyclone, Logan, UT), 10 mM HEPES (Sigma, St. Louis, MO), 1 mM sodium pyruvate (Sigma), 50 μM 2-ME (Gibco), 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma). Cells were stimulated with 1ug/ml anti-CD3 Ab and 2ug/ml anti-CD28 Ab (eBioscience) and/or 50U rmIL-2 (Peprotech) for 72 hrs. Frequency and expression level of Foxp3 were measured by flow cytometry.
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