To elucidate the effect of tumor Sdc-1 expression on the polarization of CD4+ T cells, we activated the isolated lymphocytes with plate bound anti-CD3 (10 μg/ml; clone: MEM-57), and soluble anti-CD28 (0.5 μg/ml; clone: 15E8) (Immunotools, Friesoythe, Germany), in complete RPMI 1640 medium containing 30% CM of control and Sdc-1-silenced SUM-149 cells for 96 h. The optimal concentration of CM was selected based on our pilot experiments.
Anti cd3
Manufactured by Immunotools
Sourced in Germany
Anti-CD3 is a monoclonal antibody that binds to the CD3 complex on the surface of T cells. The CD3 complex is essential for T cell activation and signal transduction. Anti-CD3 can be used in various immunological assays and applications to study T cell function and activation.
Automatically generated - may contain errors
Lab products found in correlation
Il 17 pe, by Miltenyi Biotec (2 mentions)
Ifnγ apc cytokine secretion detection kits, by Miltenyi Biotec (2 mentions)
Interleukin 2 (il 2), by Immunotools (2 mentions)
Celltrace cfse cell proliferation kit for flow cytometry, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Anti cd4, by Immunotools (1 mentions)
4 protocols using anti cd3
1
Tumor Sdc-1 Modulates CD4+ T-cell Polarization
2
Isolation and Stimulation of Th17, Th17.1 and Th1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expanded populations of Th17, Th17.1 and Th1 cells were generated by stimulating magnetically purified monocytes and CD4+ T cells at 1:5 ratio with 0.5 μg/ml antiCD3 for seven days. IL-17-PE and IFNγ-APC cytokine secretion detection kits (Miltenyi Biotech) were used to label live Th17, Th17.1 and Th1 cells. In brief, cultures were re-stimulated with Phorbol 12,13-dibutyrate (PDBu) (10 ng/ml) and ionomycin (1 nM) for 2 h before labeling with IL-17 and IFNγ catch reagents on ice at 10 × 106 cells/80 μl MACS buffer for 5 mins. Cells were transferred to pre-warmed RPMI and incubated for 40 mins at 37 °C at 4 × 105 cells/ml under continual rotation. Cells were then diluted 1:1 with ice-cold MACS buffer and chilled on ice for 10 min before centrifuging and labelling with IL-17-PE and CD3-PerCP for 15 min on ice with addition of IFNγ-APC during the final 10 min. After washing, Th17, Th17.1, Th1 and cytokine double-negative (DN) populations were collected into RPMI by FACS. Sorted T cells were then stimulated with negatively enriched (StemCell Technologies) and CD14+ FACS-purified allogenic monocytes at 1:4 ratio and 0.5 μg/ml anti-CD3 (OKT3) for 2 days in the presence of 40units/ml IL-2 (Immunotools) ± 100 nM 1,25(OH)2D3. Cell purities were >99% for Th17, Th1, DN and monocytes and >90% for Th17.1 cells.
3
Multicolor Flow Cytometry for T-Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were extracted by dissolving the microspheres in 55 mM Sodium Citrate and 10 mM EDTA in PBS for 10 min at 37°C. Cells were then suspended in PBS. Surface and intracellular staining was done in a three-colour analysis with combinations of fluorescein isothiocyanate (FITC), phycoerythrin (PE) and allophycocyanin (APC). Antibodies used included anti-CD3, anti-CD4, anti-CD8, anti-CD14 and anti-CD68 (ImmunoTools, Germany). For T–cell proliferation, PBMCs were stained with CellTrace CFSE Cell Proliferation Kit for flow cytometry (ThermoFisher Scientific, UK) before infection with Mtb. Fluorescence was then analyzed by flow cytometry (BD Accuri C6 flow cytometer).
4
Generating Distinct T Helper Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expanded populations of Th17, Th17.1 and Th1 cells were generated by stimulating magnetically purified monocytes and CD4+ T cells at 1:5 ratio with 0.5ug/ml antiCD3 for seven days. IL-17-PE and IFNγ-APC cytokine secretion detection kits (Miltenyi Biotech) were used to label live Th17, Th17.1 and Th1 cells. In brief, cultures were re-stimulated with Phorbol 12,13-dibutyrate (PDBu) (10ng/ml) and ionomycin (1nM) for 2hours before labeling with IL-17 and IFNγ catch reagents on ice at 10x10 6 cells/80μl MACS buffer for 5mins. Cells were transferred to pre-warmed RPMI and incubated for 40mins at 37°C at 4x10 5 cells/ml under continual rotation. Cells were then diluted 1:1 with ice-cold MACS buffer and chilled on ice for 10 minutes before centrifuging and labelling with IL-17-PE and CD3-PerCP for 15 minutes on ice with addition of IFNγ-APC during the final 10 minutes. After washing, Th17, Th17.1, Th1 and cytokine double-negative (DN) populations were collected into RPMI by FACS. Sorted T cells were then stimulated with negatively enriched (StemCell Technologies) and CD14+ FACS-purified allogenic monocytes at 1:4 ratio and 0.5μg/ml anti-CD3 (OKT3) for 2 days in the presence of 40units/ml IL-2 (Immunotools) ± 100nM 1,25(OH) 2 D 3 . Cell purities were >99% for Th17, Th1, DN and monocytes and >90% for Th17.1 cells.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!