For Th17 differentiation, isolated CD4+ T cells were cultured for 10 days with anti-CD3 (BioLegend Cat# 300314, RRID:AB314050 ) plus anti-CD28 mAbs (BD, Cat#555725) (at 5 μg/ml and 2 μg/ml, respectively) in the presence of the combination of cytokines and blocking antibodies appropriate for polarization: rhIL-6 h IL-1β (10 ng/ml), rhIL-23 (20 ng/ml), rhTGF-β1 (2 ng/ml), anti-IFN-γ (10 μg/ml) and anti-IL-4 (10 μg/ml). For Treg polarization, cells were cultured with TGF-β (5 ng/ml) and IL-2 (20 U/ml) (all cytokines from R&D systems) for 5 days. Where indicated hTSP-1 (5 μg/ml), mouse anti-human CD47 (1 μg/ml) clone B6H12.2 ( Abcam Cat# ab3283, RRID:AB303671 ) or IgG1 isotype control were also added. Percentage of IL-17+ and FoxP3+ CD25+ cells (Treg) was measured in a FACS Canto cytometer and analyzed with FlowJo software.
Anti cd28 mab
Manufactured by BD
Sourced in United States, Switzerland, China
Anti-CD28 mAb is a monoclonal antibody that specifically binds to the CD28 receptor on the surface of T cells. The CD28 receptor plays a crucial role in the activation and proliferation of T cells, which are essential components of the adaptive immune system.
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Lab products found in correlation
Anti cd3 mab, by BD (11 mentions)
Golgistop, by BD (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Clone 145 2c11, by BD (2 mentions)
Interleukin 2 (il 2), by Roche (2 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti cd3, by BioLegend (1 mentions)
Anti cd45 antibody, by BD (1 mentions)
Accuri c6 plus, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm fixation permeabilization solution kit, by BD (1 mentions)
Fitc labeled anti ifn γ, by BioLegend (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Anti cd3, by BD (1 mentions)
1 methyl l tryptophan, by Merck Group (1 mentions)
3h tdr, by Cytiva (1 mentions)
Fitc labeled anti ifn γ, by BD (1 mentions)
27 protocols using anti cd28 mab
1
Modulating Th17 and Treg Differentiation
2
Isolation and Culture of T Lymphocytes
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PBMCs were incubated in RPMI 1640 culture medium supplemented with 10% FBS in 6-well plates for two days at 37°C. After removing the adherent cells, the suspension cells were collected and labeled with anti-CD45 antibodies (BD Biosciences, USA; 564,106). T lymphocytes were cultured in RPMI medium supplemented with 10% fetal bovine serum, anti-CD3 mAbs (1 µg/mL) (BD Biosciences; 555,336) and anti-CD28 mAbs (2.5 µg/mL) (555,725) at 37°C for 2 h, washed, and counted with an Accuri C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA). Then, the lymphocytes were seeded in a 24-well flat-bottomed plate and cultured with RPMI medium containing IL2 (50 U/mL) for 7 days as described in a previous study [13 ]. We cocultivated T lymphocytes with HGC-27 cells transfected with the NC or miR-128-3p mimic, GES-1 cells infected with LV-NC or LV-miR-128-3p-inhibitor, conditioned medium containing recombinant IL16 or IL10 (2 µg/mL) (Novoprotein Scientific Inc., Shanghai, China; C045/CX04) or anti-CD4 blocking antibody (anti-CD4 BA) (1 µg/mL) (ab133616) at 37°C for 48 h, and these lymphocyte cells were stained and analyzed by flow cytometry [22 (link)].
3
Cytokine Production Analysis by Intracellular Staining
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Cytokine production was analyzed by intracellular staining (ICS) assay. PBMCs were incubated with or without the relevant peptides (20 μg/ml) plus anti-CD28 mAb (4 μg/ml) (BD Biosciences) and the Protein Transport Inhibitor Cocktail (Brefeldin A and Monensin; eBioscience), or with the Cell Stimulation Cocktail (phorbol 12-myristate 13-acetate [PMA], ionomycin, brefeldin A, and monensin; eBioscience) as a positive control, for 18 h at 37°C [16 (link),22 (link)]. Cells were washed and then stained with APC-labeled—HLA-A*0201 dextramers complexed to corresponding peptides, PeCy7-labeled mAb to CD8 (BioLegend), and the dump channel reagents. Cells were fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) at 4°C for 20 min, rewashed with the BD Perm/Wash buffer (BD Biosciences), and stained with different combinations of AlexaFluor700-labeled IL-17A (BioLegend) and FITC-labeled anti-IFN-γ (BioLegend) for 20 min at 4°C. Cells were washed, acquired with the LSRFortessa cytometer, and analyzed with FlowJo software. IL-17, IFN-γ or IL-17/IFN-γ producing cells were analyzed in CD8+dextramer+ cells after exclusion of B cells, monocytes, NKT cells, NK cells, and CD4+ T cells (dump channel).
4
Anti-CD3/CD28 T-cell Proliferation Assay
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For anti-CD3/CD28-induced T-cell proliferation, total T cells were isolated by negative selection (StemCell Technologies). T cells from the spleen or MxLNs were labelled by CFSE and cultured for 3 days in plates coated with anti-CD3 (1 μg ml−1) in the presence of anti-CD28 mAb (BD Bioscience, cat. no. 553295, 1 μg ml−1). Proliferation was determined with CFSE dilution.
5
Immune Cell Suppression by Tumor-Derived MSCs
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Purified CD4+ or CD8+ human T cells (105 cells) were stimulated with either 105 irradiated (9000 rad) allogeneic T-cell-depleted PBMCs (MLR – mixed lymphocytes reaction) plus 1 μg ml−1 of anti-CD3 mAb (BD Biosciences); or 5 μg ml−1 anti-CD3 plus 5 μg ml−1 anti-CD28 mAb (BD Biosciences), in the absence or presence of different numbers of tumour-MSCs (2 × 104,104,103,102 per well). Co-culture experiments were performed in RPMI 1640 medium (Seromed), supplemented as indicated in the first paragraph. On day 5, cultures were pulsed for 8 h with 0.5 μCi (0.0185 MBq) of 3H-TdR (Amersham, Little Chalfont, UK) harvested, and radionuclide uptake was measured by scintillation counting. Some experiments were conducted in the presence of the IDO1 inhibitor 1-methyl-L-tryptophan (1, 0.5, or 0.25 mM ) or in the presence of the COX inhibitor indomethacin (1, 0.5, 0.25 mcM ) purchased from Sigma Aldrich.
6
Immethridine-treated DCs Modulate T Cell Differentiation
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DCs were treated with immethridine (1 μM) and LPS (100 ng/ml) for 24h. Naïve CD4+ T cells were prepared by Mouse CD4 Negative Selection Kit (Stemcell Technology, Canada) from spleens of C57BL/6 mice and co-cultured with immethridine-treated or untreated DCs at a ratio of 10:1 for 72h at the present of 0.5 μg/ml anti-CD3 mAb and 5 μg/ml anti-CD28 mAb (BD Biosciences). The percentage of Th1 and Th17 cells was analyzed by intracellular staining with FITC-labeled anti-IFN-γ or FITC-labeled anti-IL-17A (BD Biosciences, San Jose, CA).
7
Splenic CD4+ T Cell Differentiation
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The mouse spleens were collected for cell preparation and washed twice with PBS. The spleens were minced and the red blood cells were lysed with 0.83% ammonium chloride. The cells were filtered through a cell strainer and centrifuged at 1300 rpm at 4°C for 5 min. The cell pellets were resuspended in RPMI 1640 medium and plated in 24-well plates (Corning, NY, USA) at a concentration of 1×106 cells/well. Splenic CD4+ T cells were and stimulated with 0.5 µg/ml plate-bound anti-CD3 mAb and 1 µg/ml anti-CD28 mAb (BD Pharmingen) for 3 days under Th17-polarizing condition (2 µg/ml anti-IFN-γ, 2 µg/ml anti-IL-4, 2 ng/ml transforming growth factor (TGF)- β, 20 ng/ml IL-6). After 3 days of stimulation, the cells were restimulated with 25 ng/ml PMA and 250 ng/ml ionomycin (both from Sigma, St. Louis, MO) in the presence of GolgiStop (BD Pharmingen) for 5 h.
8
Proliferation and Intracellular Cytokine Assays for T. cruzi Antigens
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For proliferation assays, splenocytes were stained with CellTrace Violet (ThermoFischer, USA), following the manufacturer’s instructions. For intracellular assays, cells were maintained in culture with Brefeldin A (3 µg/ml) and Golgistop (1/2,000, final dilution) (BD Biosciences, USA). Cultures containing 1 × 106 cells were kept in RPMI 1640 supplemented with 10% FCS, 2mM l -glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 µM 2-mercaptoethanol for 72 h (proliferation assays) or 12 h (intracellular cytokine staining assays) at 37°C and 5% CO2 atmosphere in the presence of T. cruzi antigen (50 µg/ml), anti-CD3 mAb (10 µg/ml; clone 145-2C11), and anti-CD28 mAb (2 µg/ml; clone 37.51) (BD Biosciences, USA), or media. T. cruzi Sylvio X10/4 antigen was produced by pelleting parasites obtained from supernatant of LLC-MK2 cell cultures, followed by 20 freeze–thawing cycles and centrifugation at 3,000 rpm to eliminate small clumps. Antigen was stored at 2.5 mg/ml in −80°C and optimal final concentration for in vitro lymphocyte stimulation was determined.
9
Activation Induced Molecule Assay for HIV Gag
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Cryopreserved PBMC were thawed and allowed to rest for 2–3 hours prior to stimulation with HIV gag PTE peptides for a modified AIM (Activation Induced Molecule) assay [58 (link)]. Cells were cultured at 5 million per milliliter for 18–20 h in presence of 2 μg/ml GAG PTE peptides (AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases [NIAID], National Institutes of Health [NIH]: HIV-1 PTE Gag Peptide Pool from NIAID, Division of AIDS), 1 μg/ml of staphylococcal enterotoxin B (SEB; List Biological Laboratories) or medium (negative control). At the beginning of culture, the following reagents were added: 1 μg/ml costimulatory molecule anti-CD28 mAb (BD Biosciences), 1ug/mL of anti-CD49d (BD Biosciences) and 0.5ug/mL the degranulation marker (CD107a PerCP-Cy5.5).
Following the culture period, cells were stained for flow sorting using previously titrated mAb (CD3, CD4, CD8, OX40, and CD25). Sorting was performed using the gating strategy shown (S5 Fig
Following the culture period, cells were stained for flow sorting using previously titrated mAb (CD3, CD4, CD8, OX40, and CD25). Sorting was performed using the gating strategy shown (
10
Activation of Human T Cells by BiTE
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We used the standard Ficoll (GE Healthcare, Pittsburgh, PA, USA) density gradient centrifugation procedure to isolate human PBMCs from buffy coats provided by healthy donors from the First Affiliated Hospital of Soochow University (Suzhou, China). The harvested PBMCs were washed with PBS (pH 7.4) and resuspended in RPMI-1640 (SH30027.01) cell medium containing 10% FBS (SH30401.01). Cell culture for PBMCs was carried out as previously described.
On day 1, 100 μL cells were plated onto a 24-well plate at a density of 3 × 106 cells/mL per well. BiTE-hIgFc (STL001), a mixture of aCD138-ScFv-hIgFc and aCD3-ScFv-hIgFc, and isotype control human IgG1-Fc (Abcam, Cambridge, UK) were diluted to 100 ng/mL with PBS (pH 7.2). Anti-CD28 mAb (100 ng/mL; BD Biosciences) and IL-2 (10 U/mL; Roche, Basel, Switzerland) were added to different groups of the T cell activation assay. After T cell activation for 24 h, the CD4+ or CD8+ T cells were gated, and the activated T cell surface markers CD25 and CD69 were analyzed using anti-CD25 and anti-CD69 antibodies (BD Biosciences) by a flow cytometer (BD Biosciences).
On day 1, 100 μL cells were plated onto a 24-well plate at a density of 3 × 106 cells/mL per well. BiTE-hIgFc (STL001), a mixture of aCD138-ScFv-hIgFc and aCD3-ScFv-hIgFc, and isotype control human IgG1-Fc (Abcam, Cambridge, UK) were diluted to 100 ng/mL with PBS (pH 7.2). Anti-CD28 mAb (100 ng/mL; BD Biosciences) and IL-2 (10 U/mL; Roche, Basel, Switzerland) were added to different groups of the T cell activation assay. After T cell activation for 24 h, the CD4+ or CD8+ T cells were gated, and the activated T cell surface markers CD25 and CD69 were analyzed using anti-CD25 and anti-CD69 antibodies (BD Biosciences) by a flow cytometer (BD Biosciences).
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