The slowly hydrolyzable ATP analog ATPγS [7 (link), 20 (link)] (Sigma Aldrich, St. Louis, MO, USA) as well as the suramin analog NF340 [7 (link), 10 (link), 13 (link), 21 (link)] (Santa Cruz, Dallas, TX, USA) served as P2Y11 receptor agonist (20 µM) and antagonist (20 µM), respectively. Further reagents utilized in this study include the PDE4-selective inhibitor rolipram (10 µM) (Sigma-Aldrich), BAPTA-AM (10 µM) (Sigma-Aldrich), calphostin C (250 nM) (Tocris), recombinant IL-1α and IL-1β (0.5–2 ng·ml−1) (R&D Systems, Minneapolis, MN, USA), the TACE/ADAM17 inhibitor TAPI-1 (20 µM) (Tocris), the selective inhibitor of Epac1 (R)-CE3F4 (20 µM) (Tocris), and the humanized anti-VEGF monoclonal antibody bevacizumab (0.5 µg ml−1) (Selleckchem, Houston, TX, USA).
Tapi 1
Manufactured by Bio-Techne
Sourced in United States
TAPI-1 is a lab equipment product offered by Bio-Techne. It functions as a potent and selective inhibitor of TACE (TNF-alpha converting enzyme), also known as ADAM17.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii fortessa, by BD (2 mentions)
Il 18, by BioLegend (2 mentions)
Facsymphony a5, by BD (2 mentions)
Fitc labeled mouse anti human cd107a clone h4a3, by BD (2 mentions)
Il 12, by BioLegend (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Rituximab, by Roche (2 mentions)
Rolipram, by Merck Group (1 mentions)
Bevacizumab, by Selleck Chemicals (1 mentions)
Calphostin c, by Bio-Techne (1 mentions)
Atpγs, by Merck Group (1 mentions)
Nf340, by Santa Cruz Biotechnology (1 mentions)
Bapta am, by Merck Group (1 mentions)
Gi254023x, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Facsymphony a5 flow cytometer, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Amplex red, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
6 protocols using tapi 1
1
Modulation of P2Y11 Receptor Activity
2
ADAM10 and ADAM17 Inhibitor Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For ADAM10 and ADAM17 inhibitor studies, 5.0 × 105 cells were seeded in 1 mL of DMEM without supplements per well of a 24‐well plate dish. Cells were treated with indicated concentrations of phorbol 12‐myristate 13‐acetate (PMA; Sigma‐Aldrich, Steinheim, Germany) diluted in equal volumes of DMSO (Sigma‐Aldrich) 30 min prior to treatment with 2 µm of ADAM10 inhibitor GI254023X (Sigma‐Aldrich) or 5 µm of ADAM17 inhibitor TAPI‐1 (Tocris, Minneapolis, MN, USA). DMSO‐treated cells served as negative controls. After 48 h of incubation, cell culture supernatant was collected and centrifuged at 10 000 g for 10 min at 4 °C. Equal volumes of cell‐free supernatant were analyzed with regard to soluble L1CAM by western blot analysis as described in previously.
3
Cytotoxicity Assay for NK and ADCC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cell suspensions of liver and spleen tissue were cocultured with K562 or Raji cells at a ratio of 10:1 in RPMI 1640 (GIBCO, Thermo Fisher Scientific) with 10% FCS in a humidified incubator at 37°C and 5% CO2. Culture medium contained FITC-labeled mouse anti-human CD107a (clone H4A3, BD Bioscience) antibody and monensin was added after 1 hour of coculture. For the ADCC assay, Raji cells were incubated with Rituximab (MabThera, Roche) at a concentration of μg/ml for 10min at 37°C prior to the addition of effector cells. For the stimulation with IL-12 (20ng/ml, Biolegend) and IL-18 (10ng/ml, Biolegend), the culture medium was supplemented with metalloproteinase inhibitor TAPI-1 (5 μg/ml, Tocris), and monensin and Brefeldin A was added after 1 hour of stimulation. After a total of 5 hours, cells were washed, stained, and analyzed on a BD LSR II Fortessa or BD FACSymphony A5 flow cytometers as described above.
4
NK Cell Degranulation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For degranulation assays, preactivated human NK cells were cocultured with human melanoma cells at an effector to target cell ratio of 1:1 in complete RPMI 1640 containing PE-labeled anti-human CD107a antibody (H4A3, BioLegend) in a humidified incubator at 37°C and 5% CO2. For KIR-matched assays, culture medium was supplemented with the metalloproteinase inhibitor TAPI-1 (5 μg/ml; Tocris) to prevent shedding of the CD16 molecule following NK cell activation. Brefeldin A (5 μg/ml; Sigma-Aldrich) and monensin (2 μM; Thermo Fisher Scientific) were added after 1 hour of incubation. After a total of 5 hours of incubation, cells were washed, stained, and analyzed on a BD FACSymphony A5 flow cytometer. UMAP algorithm (81 ) visualization was performed using FlowJo software (version 10, TreeStar Inc.). Data were pregated on live, CD45+ cell populations, downsampled to 27,000 cells per condition using the DownSample (v3.3) plugin, and subsequently concatenated per donor. The UMAP plugin (v3.1) for FlowJo was used with the following parameters for dimensionality reduction: “Euclidean,” nearest neighbors: 50; minimum distance: 0.25; number of components: 2.
5
Cytotoxicity Assay for NK and ADCC
6
Histamine Receptor Agonists and EGFR Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histamine and the specific histamine receptor agonists 2-([3-trifluoromethyl]-phenyl histamine dimaleate (H1R agonist), amthamine dihydrobromide (H2R agonist), and (R)α-methylhistamine dihydrochloride (H3R agonist) were from Sigma-Aldrich Corp. (St. Louis, MO, USA). The receptor agonist 4-methylhistamine dihydrochloride (H4) and TAPI-1 (ADAM17 inhibitor) were from Tocris Bioscience (Ellisville, MO, USA). EGF was from Sigma-Aldrich, and Amplex Red and fura-2-acetoxymethyl ester (fura-2/AM) were purchased from Invitrogen (Grand Island, NY, USA). An EGFR antagonist was from LC Laboratories (Tyrphostin AG 1478; Woburn, MA, USA). Antibodies directed against the EGFR, phosphorylated EGFR (Tyr1068), AKT, phosphorylated AKT, and Western blotting application solution kits were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against ERK and phosphorylated ERK and small interfering RNA (siRNA) transfection medium were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transfection reagent, rat EGFR siRNA–SMART pool, and nontargeting pool were from Dharmacon RNAi Technology (DharmaFECT1, ON-TARGETplus, ON-TARGETplus, respectively; Lafayette, CO, 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!