Canine NK cells were isolated using previously described methods [19 (link)]. Peripheral blood mononuclear cells (PBMCs) were isolated from a beagle dog (Genia Inc., Korea). Subsequently, CD5 negative (CD5lo) cells were isolated by immunomagnetic separation and cultured in a cell culture flask at 37°C in a 5% CO2 incubator supplemented with 500 U/mL human IL-2, 10 ng/mL human IL-15, and 5 ng/mL canine IL-21 (all from R&D System, Minneapolis, USA). After 21 days, cell surface markers were analyzed via FACS flow cytometry of activated CD5lo cells. The activated canine NK cell markers were identified by labeling, CD5lo cells (1 × 105) with antibodies against surface markers including mouse anti-dog CD3-FITC (clone CA17.2A12, Bio-Rad, Hercules, USA), rat anti-dog CD4-FITC (clone YKIX302.9, Bio-Rad), rat anti-dog CD5-PE (clone YKIX322.3, Bio-Rad), mouse anti-dog CD21-APC (clone CA2.1D6, Bio-Rad), rat anti-dog CD45-APC (clone YKIX716.13, Bio-Rad), and rat anti-dog major histocompatibility complex (MHC)-II-FITC (clone YKIX334.2, eBioscience, San Diego, USA) for 1 hr. The labeled cells were washed twice with phosphate buffered saline and analyzed using a FACSCalibur™ flow cytometer (Becton Dickinson, USA) with Cell Quest Pro software (Becton Dickinson) for data analysis.
Human il 2
Manufactured by R&D Systems
Sourced in United States
Human IL-2 is a recombinant protein that mimics the structure and function of the naturally occurring human interleukin-2 cytokine. It is commonly used in cell culture and research applications to study immune system function and T-cell activation.
Automatically generated - may contain errors
Lab products found in correlation
Human il 15, by R&D Systems (2 mentions)
Mouse il 2, by R&D Systems (2 mentions)
Cd3 cd28 dynabead, by Thermo Fisher Scientific (2 mentions)
Facsaria sorter, by BD (2 mentions)
Streptavidin conjugated microbeads, by Miltenyi Biotec (2 mentions)
Biotinylated anti cd8 and ter119 antibodies, by Thermo Fisher Scientific (2 mentions)
Mouse il4, by Cytek Biosciences (2 mentions)
Lsr 2 cytometer, by BD (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by GE Healthcare (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Anti cd3 cd28 beads, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Cfda se, by BD (2 mentions)
Human il 21, by R&D Systems (2 mentions)
Imdm medium, by Thermo Fisher Scientific (2 mentions)
Cpg odn 2006, by InvivoGen (2 mentions)
Human il 4, by R&D Systems (2 mentions)
Human recombinant hcd40l, by R&D Systems (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
33 protocols using human il 2
1
Isolation and Characterization of Canine NK Cells
2
Activated Primary T Cell Proliferation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Assays with human primary T cells were conducted by Pharmaron. Human peripheral blood mononuclear cells for these assays were sourced commercially from Sailybio (Cat. No. SLB-HP200A), with ethical approvals and informed consent for the collection. Human primary T cells were isolated from fresh peripheral blood mononuclear cells (Sailybio) using the human Pan T Cell Isolation Kit (Miltenyi Biotec) and resuspended in RPMI medium 1640 containing 10% FBS, 1% penicillin/streptomycin, and 10 ng/mL human IL-2 (R&D Systems). Mouse primary T cells were isolated from fresh spleen cells using the mouse Pan T Cell Isolation Kit (Miltenyi Biotec) and resuspended in RPMI medium 1640 containing 10% FBS, 1% penicillin/streptomycin, and 10 ng/mL mouse IL-2 (R&D Systems). Compounds R80 and T35 were added as appropriate to various concentrations. Cells were seeded into 96-well plates and incubated at 37 °C and 5% CO2 for 1 h. Human or mouse anti-CD3/anti-CD28 Dynabeads (Thermo Fisher Scientific) (78 (link)) and 100 μM cytidine (where appropriate) were then added, and cells were incubated at 37 °C and 5% CO2 for 5 d. CTG reagent (Promega) was added to cells and incubated at room temperature for 30 min, after which luminescence was recorded using a Perkin-Elmer Envision microplate reader.
3
PBMC-Based IFN-γ Production Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs (1 × 106 cells) from cynomolgus macaques challenged with v7D subcutaneously were incubated with 2000 PFU of cell-free rOka virus, or MRC-5 cell lysates as a negative control, along with 20 U/mL human IL-2 (R&D Systems). Half of the medium was changed every two days, and on day 5, quantitation of IFN-γ concentrations in the culture supernatants was performed with a commercial ELISA kit (Mabtech) specific for nonhuman primate IFN-γ per the manufacturer’s instructions.
4
T cell functional assay for NASH-HCC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For T cell coculture assay, T cells were isolated by the EasySepTM Mouse T cell Isolation Kit (STEMCELL). Isolated T cells were cultured with conditional medium collecting from Hepa1-6 cells, supplied with mouse T-activator CD3/CD28 Dynabeads (Invitrogen) and mouse IL-2 (Biolegend, 10ng/ml) for 72 hours. Human peripheral blood CD8+ T cells were obtained from STEMCELL (#70027). Human CD8+ T cells were co-cultured with conditional medium collecting from different NASH-HCC cells, with human T-activation CD3/CD28 Dynabeads (Invitrogen) and human IL-2 (R&D Systems, 10ng/ml) for 72 hours, with or without cholesterol (Sigma), cholesterol ester (Sigma) or β- cyclodextrin (Sigma). Quantification of IFN-γ + and GZMB+ CD8+ T cells (among CD8+ T cells) from different groups was conducted. T cells were stimulated with PMA, monensin, and ionomycin for 4 hours. IFN-γ or GZMB-producing cells were determined by flow cytometry.
5
Engineered T Cell Cytotoxicity Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cytotoxicity of engineered T cells was evaluated using the standard lactate dehydrogenase release assay (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega) following the manufacturer’s recommendations. The 5 × 103 BT-474 cells were cocultured with Mock T or BsCAR T cells for 12 to 16 h and in the presence of 10 to 0.016 nM or 1 nM in case of E:T titration of DARPins-Bn in RPMI (Gibco) media supplemented with 40 U/mL human IL-2 (R&D Systems). To assess the basal levels of BsCAR T and mock T cell cytotoxicity, the T cells were incubated in the absence of drug conjugates. The maximum cell lysis was determined by target cell lysis using 10% (vol/vol) cell lysis solution. The following formula was used to calculate the release of lactate dehydrogenase: cytotoxicity = 100 × [((CAR T cell + target cell + DARPin-Bn) − (CAR T cell + target cell))/(max target cell lysis − target cells alone)]. The IC50 value was determined by GraphPad Prism software.
6
Activation of Primary Human T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary human T cells from healthy donors were obtained from BioIVT (Colmar, PA) and were activated by Human T-Activator Dynabeads (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions and in the presence of human IL-2 (10 ng/ml) (R&D Systems) in complete T cell media (RPMI 1640 media containing 10% fetal bovine serum and 1% GlutaMAX) according to the manufacturer’s instructions. After 3 days of stimulation, the anti-CD3/CD28 beads were removed, and cells were maintained in IL-2 containing human T cell complete media [IL-2 (20 ng/ml)] for up to 4 days before experimentation.
7
Expansion of Mart-1-Specific CD8+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
Drosophila APC line 668 was treated with UVADEX as described above. The APCs were then loaded with 10 μM of Mart-1 peptide at room temperature for 4 h. Human CD8 T cells were purified from HLA-A2 positive PBMC and cultured with Mart-1-peptide-loaded APCs or UVADEX-treated Mart-1-peptide-loaded APCs at 37°C, 5% CO2 for 5 days. Human IL-2 (20 U/ml, R&D) and IL-7 (30 U/ml, R&D) were added at day 5 for further culture. The activated CD8 T cells were restimulated twice at day 7 and day 15 with non-CD8 adherent PBMCs from the same donor in the presence of Mart-1 peptide. The number of antigen-specific CD8 T cells was identified by Mart-1/HLA-A2 tetramer (Beckman Coulter, CA) staining at day 19.
+ Expand
Drosophila APC line 668 was treated with UVADEX as described above. The APCs were then loaded with 10 μM of Mart-1 peptide at room temperature for 4 h. Human CD8 T cells were purified from HLA-A2 positive PBMC and cultured with Mart-1-peptide-loaded APCs or UVADEX-treated Mart-1-peptide-loaded APCs at 37°C, 5% CO2 for 5 days. Human IL-2 (20 U/ml, R&D) and IL-7 (30 U/ml, R&D) were added at day 5 for further culture. The activated CD8 T cells were restimulated twice at day 7 and day 15 with non-CD8 adherent PBMCs from the same donor in the presence of Mart-1 peptide. The number of antigen-specific CD8 T cells was identified by Mart-1/HLA-A2 tetramer (Beckman Coulter, CA) staining at day 19.
8
Ex Vivo Expansion of Regulatory T Cells
9
Proliferation Assay for iNKT Cells in LDLR Mutations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs of patients with LDLR mutations and age-matched controls were subjected to a standard proliferation assay (52 (link)). In short, PBMCs were incubated with 10% heat-inactivated human AB serum, 100 ng/mL αGalCer, and 100 IU/mL human IL-2 (R&D Systems, Bio-Techne) in standard T cell medium. On day 0, baseline APC and iNKT cell phenotypes were assessed using flow cytometry. On day 14, iNKT cell proliferation and phenotype were assessed using flow cytometry. Cells were stained with a viability dye (BioLegend) and mAbs specific for CD3 (HIT3a), CD4 (L200), CD8 (RPA-T8), CD25 (2A3), CD69 (FN50), and CD1d tetramer (NIH) for iNKT cell phenotyping (from BioLegend, except for CD1d).
10
Recombinant Protein Expression in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The E. coli strain JM-109 (Promega) was used for plasmid proliferation and cloning. The E. coli strain Rosetta-gami 2 (Novagen) was used as a host for the production of recombinant proteins. Human nasopharyngeal carcinoma cell line CNE2 and human embryonic kidney cell line (HEK 293T) were purchased from the American Type Culture Collection (ATCC). Plasmid pET-44a was purchased from Promega. All primers used in this study were synthesized by Biosune Biology and Technology Company (Shanghai, China). NdeI and XhoI restriction endonucleases, T4 DNA ligase, and PrimeSTAR HS DNA polymerase were purchased from Takara Biotechnology (Dalian, China). Human IL-2, IFN-γ, and TNF-α enzyme linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, Minnesota, USA). β-tubulin rabbit mAb and EGFR rabbit mAb were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Strep-Tag II mAb was purchased from Merck (Billerica, Massachusetts, USA). (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) (MTS) reagent for cell proliferation assay was purchased from Promega (Madison, Wisconsin, USA). Annexin V-conjugated Alexa Fluor 488 (Alexa488) Apoptosis Detection Kit was purchased from BD Pharmingen (San Diego, California, USA).
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!