Peripheral blood mononuclear cells were prepared from buffy coats obtained from healthy donors by centrifugation through Ficoll-Paque (GE healthcare, Uppsala, Sweden). CD4+ T cells were isolated by magnetic bead depletion of CD8, CD14+, CD15+, CD16+, CD19+, CD36+, CD56+, CD123+, T cell receptor-gamma/delta, and glycophorin A–positive cells (CD4+ T Cell Isolation Kit) on an AutoMACS instrument (Miltenyi Biotec). CD4+ T cells (5×105 cells/well) were cultured with X-VIVO medium (Lonza, Walkersville, MD) containing 2% FBS and 5 µg/ml anti-CD28 antibody (clone CD28.2, BioLegend, San Diego, CA) in anti-CD3-precoated 24-well culture plates (clone OKT3, BioLegend). During in vitro proliferation of CD4+ T cells, human amnion-, chorion-, or bone marrow-derived (Lonza) MSCs were co-cultured at 5×104 cells/well. After 5 days of co-culturing, T cells were separated from the monolayer MSCs and counted with an automated cell counter (Countess, Invitrogen).
Anti cd28 antibody clone cd28
Manufactured by BioLegend
Sourced in United States
The Anti-CD28 antibody (clone CD28.2) is a laboratory reagent used to stimulate and activate T cells. It binds to the CD28 receptor on the surface of T cells, which is a key co-stimulatory molecule involved in T cell activation and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Automacs instrument, by Miltenyi Biotec (1 mentions)
Countess, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
X vivo medium, by Lonza (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Staphylococcal enterotoxin b, by Merck Group (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Anti cd3 antibody clone okt3, by BioLegend (1 mentions)
Interleukin 2 (il 2), by BioLegend (1 mentions)
Recombinant human il 2, by BioLegend (1 mentions)
Anti cd3 antibody clone okt, by BioLegend (1 mentions)
4 protocols using anti cd28 antibody clone cd28
1
Mesenchymal Stem Cell Modulation of CD4+ T Cell Proliferation
2
SARS-CoV-2 Epitope Mapping in Human PBMCs
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PBMCs (2 × 106) were stimulated with peptide pools of individual proteins of SARS-CoV-2 (JPT Peptide Technologies; i.e., NCAP, spike S1, spike S2, VEMP, VME1, AP3a, NS6, NS7a, NS7b, NS8, ORF9b, ORF10, Y14) at 1 μg/mL each in the presence of 1 μg/mL purified anti-CD28 antibody (clone CD28.2, BioLegend). Unstimulated controls were supplemented with equal concentrations of DMSO and 4 mg/mL staphylococcal enterotoxin B (SEB) (Sigma-Aldrich), and CMV peptide pool (pp65 and IE-1; 0.5 μg/mL each; JPT Peptide Technologies) served as positive controls. Stimulated PBMCs were incubated in a humidified incubator at 37°C, 5% CO2 for 16 h. Intracellular cytokine production was captured by addition of 2 μg/mL Brefeldin A (Sigma-Aldrich) after 2 h of stimulation, and cells were stained using antibodies (all from BioLegend) and the FoxP3/Transcription Factor Staining Buffer Set (eBioscience). Staining was performed using fluorophore-conjugated human anti-CD3 (OKT3), -CD4 (SK3), -CD8 (RPA-T8), -IFN-γ (4S.B3), -TNF-α (MAb11), -IL-2 (MQ1-17H12), -CD137 (4B4-1), -CD154 (24–31), -CCR7 (G043H7), and -CD45RA (HI100) antibodies. LIVE/DEAD Fixable Blue Dead Cell Stain (L/D; Invitrogen) was used to exclude dead cells. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter) and FlowJo-10 software (Tree Star).
3
Activation of Naïve CD8+ T Cells
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Prior to co-culture experiments, activation of isolated naïve CD8+ T cells was performed. For this purpose, a 24-well plate was coated by incubation for 2 h at 37°C with 200 µl of 1.5 µg/ml anti-CD3 antibody (clone: OKT3, Biolegend) in PBS. Before T cells were seeded, wells were washed 2-times with PBS. Then, 1.3 – 1.8x106 naïve CD8+ T cells were seeded in 1 ml TCM supplemented with 1.5 µg/ml anti-CD28 antibody (clone: CD28.2, Biolegend) and 60 ng/ml IL-2 (Biolegend). CD8+ T cells were cultured for 3 days at 37°C, 5% CO2 and 86% humidity.
4
Activation of Naïve CD8+ T Cells
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Activation of primary naïve CD8+ T cells was performed by stimulation with anti-CD3 and anti-CD28 antibodies which were used to mimic antigen-presenting cells. For this purpose, a 24-well plate was coated with 1.5 µg/ml anti-CD3 antibody (clone: OKT3, BioLegend, San Diego, CA, USA) diluted in 200 µl sterile PBS for 2 h at 37°C. Afterwards, the plate was washed twice with PBS to remove all unbound antibodies. Then, 1.5 x106 CD8+ T cells per well were seeded in 1 ml PDAC cell medium further supplemented with 2% HEPES and 1% Penicillin and Streptomycin (TCM). Finally, 1.5 µg/ml anti-CD28 antibody (clone: CD28.2, BioLegend, San Diego, CA, USA), and 60 ng/ml recombinant human IL-2 (BioLegend, San Diego, CA, USA) were added. After 72 h, CD8+ T cells were collected and used for subsequent coculture experiments.
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