For Th0 maintenance, 10 μg/ml anti-IL-4 (Abcam, Cambridge, MA, USA) and 10 μg/ml anti-IFN-γ (R&D system, Minneapolis, MN, USA) antibodies were added to the culture system; for Th1 induction, 10 ng/ml IL-12 (PreproTech), 10 ng/ml IL-2 (PreproTech), and 10 μg/ml anti-IL-4 antibody were added to the culture system; for Th2 induction, 10 ng/ml IL-4 (PreproTech), 5 ng/ml IL-2, 10 μg/ml anti-IL-12 (Biolegend), and 10 μg/ml anti-IFN-γ were added to the culture system; for Treg induction, 5 ng/ml IL-2 and 5 ng/ml TGF-β (PreproTech) were added to the culture system. T cells were all specifically activated by CD3/CD28 antibodies (BD biosciences).
Cd3 cd28 antibodies
Manufactured by BD
Sourced in United States
CD3/CD28 antibodies are laboratory reagents used to activate and expand T cells in in vitro cell culture systems. They mimic the natural activation of T cells by binding to the CD3 and CD28 receptors on the T cell surface.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Facsaria iiu, by BD (2 mentions)
Anti ifn γ, by R&D Systems (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Anti ifn γ, by Thermo Fisher Scientific (1 mentions)
Anti il 4, by Thermo Fisher Scientific (1 mentions)
Anti il 12, by BioLegend (1 mentions)
Anti il 4, by Abcam (1 mentions)
Il 12, by Thermo Fisher Scientific (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by BioLegend (1 mentions)
Zm241385, by MedChemExpress (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
4 protocols using cd3 cd28 antibodies
1
T Cell Polarization Protocol
2
Tregs Modulate Neutrophil Functions
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As previously described (24 (link)), Tregs (5×104 per well) were plated in 48-well plates pre-coated with CD3/CD28 antibodies (BD Bioscience) in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM of l-glutamine (Invitrogen), and 1% penicillin-streptomycin (Sigma-Aldrich). Then, neutrophils (5×105 per well) were added to the co-culture at 37°C. After 5h, neutrophil phagocytosis and oxidative bursts were measured by flow cytometry analysis (see below). In another set of experiments, ZM241385 (1μM; MedChem Express) was added as described in previous research (26 (link)).
3
Isolation and Culture of Regulatory T Cells
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The splenocytes were isolated by passing spleen tissue through a cell strainer with a mesh size of 70 nm using the back of a sterile 3 mL syringe plunger. With the aid of red blood cell lysis buffer, RBCs were successfully removed. Single splenocytes were centrifugated at 300 ×g for 10 min at 4°C and washed twice with PBS. One milliliter of cold PBS containing 2% fetal bovine serum was used to suspend the pellet. Then, the cell suspension was incubated with a CD16/CD32 antibody to block the Fc receptors, and stained with CD4-PerCP and CD25-PE antibodies. For cell co-culture experiments, CD4+CD25+ Tregs were isolated by FACS using a FACSAria IIu instrument (BD Bioscience). The purity of Treg populations assessed by 2-color flow cytometry was >95%. As previously described (24 (link)), Tregs (5×104-per well) were plated in 48-well plates precoated with CD3/CD28 antibodies (BD Bioscience) in RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin (Sigma-Aldrich), and 2 mM of l-glutamine (Invitrogen, Carlsbad, CA, USA). Treg supernatant was collected after 24 h and cultured for TGF-β and IL-10 examination.
4
Isolation and Culture of Regulatory T Cells
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Splenocytes were isolated by passing spleen tissue through a cell strainer (70 nm) using the back of a sterile syringe plunger. Red blood cell (RBC) lysis buffer was used to extract RBCs. Single splenocytes were centrifuged (300g, 10 min, 4°C) and washed twice with phosphate-buffered saline (PBS). One milliliter of cold PBS containing 2% fetal bovine serum was used to suspend the pellet. Then, the cell suspension was incubated with a CD16/CD32 antibody to block Fc receptors and stained with CD4-PerCP and CD25-PE antibodies. For cell co-culture experiments, CD4+CD25+ T regs were isolated by FACS using a cell sorter (FACSAria IIu; BD Biosciences). The purity of T reg populations assessed by two-color flow cytometry was >95%. As described previously ( 14), T regs (5 × 10 4 per well) were plated in 48-well plates precoated with CD3/CD28 antibodies (BD Biosciences) in RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin (MilliporeSigma, Burlington, MA), and L-glutamine (2 mM; Invitrogen, Carlsbad, CA). T reg supernatant was collected after 24 h and cultured for measurement of levels of transforming growth factor (TGF)-β and interleukin (IL)-10.
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