The mice were intraperitoneally (i.p.) immunized with 20 μg (in 100 μL) of chicken OVAlbumin (OVA) (Sigma-Aldrich, St. Louis, MO, USA) emulsified in alum [2.25 mg of Al(OH)3/2 mg Mg(OH)2] (Thermo Fisher Scientific, Waltham, MA, USA) on days 0 and 14 (OVA/Alum model group). On days 24, 25, and 26, the OVA/Alum mice were challenged with an aerosol of 1% OVA in saline for 40 min via ultrasonic nebulization (DeVilbiss, Somerset, PA, USA). The mice in the OVA/Alum-ABT-199 or OVA/Alum-Nf-ABT-199 groups were intratracheally (i.t.) administered different doses of ABT-199 (Selleck, Houston, TX, USA) or Nf-ABT-199 in 50 mL of vehicle 2 h after each OVA challenge. ABT-199 was formulated in 60% phosal 50 propylene glycol, 30% polyethylene glycol 400, and 10% ethanol [24 (link),25 ]. The naive mice were sensitized and challenged with saline alone at the same time. After the last allergen challenge for 24 h, all mice were sacrificed for analysis. Bronchoalveolar lavage (BAL) fluid and lungs were collected. The OVA/Alum model was established using the eosinophilic airway inflammatory model, as previously reported [26 (link)].
Abt 199
Manufactured by Selleck Chemicals
Sourced in United States, Germany
ABT-199 is a small molecule inhibitor that targets the BCL-2 protein. It is primarily used for research purposes in the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Abt 263, by Selleck Chemicals (30 mentions)
Abt 737, by Selleck Chemicals (15 mentions)
A 1331852, by Selleck Chemicals (12 mentions)
A 1210477, by Selleck Chemicals (10 mentions)
S63845, by Selleck Chemicals (9 mentions)
A 1155463, by Selleck Chemicals (8 mentions)
Ibrutinib, by Selleck Chemicals (7 mentions)
Wehi 539, by Selleck Chemicals (6 mentions)
Obatoclax, by Selleck Chemicals (5 mentions)
Cycloheximide (chx), by Merck Group (5 mentions)
Trametinib, by Selleck Chemicals (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Azd5991, by Selleck Chemicals (3 mentions)
A 1331852, by Abbvie (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Mcl 1, by Cell Signaling Technology (3 mentions)
A 1155463, by Chemietek (3 mentions)
Fumonisin b1, by Cayman Chemical (3 mentions)
Celltiter glo assay, by Promega (2 mentions)
109 protocols using abt 199
1
Ovalbumin-Induced Allergic Airway Inflammation
2
Generation of ABT199-resistant MV4-11 Cell Line
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The generation of the MV4-11 ABT199 resistant line was performed by culturing MV4-11 cells in increasing concentrations of ABT199 (Selleckchem, Radnor, TX, USA, S8048) starting from 1 nM to 100 nM for eight weeks. The cells were first cultured with an initial sub-toxic dose of 1/10th of the baseline IC50 of the cells until significant resistance was reached and cell viability of more than 90% was maintained by the cells with continuous exposures of ABT199 of up to 100 nM, which was 5-fold the IC50 of the parental MV4-11 cell line. The cells were then plated in methylcellulose-based semi-solid media in ABT199-containing medium at a density of 1000 cells/cm3 dish (MethoCult™ H4230; Stemcell Technologies, Inc., Vancouver, BC, Canada). The resistant colony was isolated using a light microscope and then cultured using regular RPMI media. This colony was named ABT199-R.
3
Cell Viability Assay with BZB and ABT-199
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Cell viability was assessed using CellTiter-Glo Assay (Promega, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, 4000 cells per well were seeded in white 96-well plates with plastic bottom (Invitrogen, Karlsbad, Germany). After 2 h BZB (0.5–10 nM; Selleckchem, Munich, Germany) and ABT-199 (1.5–30 μM; Selleckchem) were added. Cell viability was analyzed in quadruples after 48 h.
4
Culturing Pancreatic Cancer Cell Lines
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Human PDAC cell lines AsPC1, BxPC-3, CFPAC-1, HPAC, PANC1 and SW1990 were all preserved in Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University and normal human pancreatic ductal cell line hTERT-HPNE was purchased from American Type Culture Collection (ATCC, Manassas, VA). All of these cells were cultured in indicated medium according to ATCC protocols, and supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotics (100 μg/ml streptomycin and 100 units/ml penicillin) at 37°C in a humidified incubator under 5% CO2 condition. Human umbilical vein endothelial cells (HUVECs) were a generous gift from Dr. Huan Yi (Shanghai No.5 People's Hospital, Fudan University). ABT-199 was purchased from Selleck (Shanghai, China).
5
Evaluating Anti-Apoptotic Proteins in Hematological Malignancies
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All chemicals apart from ABT-199, A1331852, A1155463, A1210477 (Selleck Chemicals, Houston, TX, USA), and S63845 (ApexBio, Taiwan) were from Sigma (Deisenhofen, Germany). Most cell lines used in this study were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany) except Pfeiffer and SUDHL2 cells (American Type Culture Collection; Manassas, VA, USA), OCI-LY10 (Sandeep Dave, Duke University, Durham, NC, USA), MedB116 (link) (Peter Moeller, University of Ulm, Ulm, Germany) and Karpas-110617 (link) (Abraham Karpas, University of Cambridge, Cambridge, UK). All cell lines were authenticated by short tandem repeat profiling and routinely tested for mycoplasma contamination. Primary patient-derived samples were obtained from patients attending the University Hospital of Leicester, UK. Local ethical approval (Leicestershire, Northamptonshire and Rutland REC06/Q2501/122) and patients’ consent were obtained through the Haematological Tissue Bank of the Ernest and Helen Scott Haematological Research Institute, Leicester, UK. Peripheral blood mononuclear cells were isolated from the blood of patients presenting in leukemic phase and the CellTiterGlo assay (Promega, Mannheim, Germany) was used to assess these cells’ viability.
6
Evaluating AML Cell Sensitivity to BCL-2 Inhibitors
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The sensitivity of AML cell lines towards ABT-199 (Selleck), A1331852 (Selleck) and S63845 (Appexbio) was assessed with CellTiter-Glo viability assay (Promega). Cells were seeded in a density of 1 × 105 cells/ml in a white 96-well plate. After 72 h of incubation 5 μl/well of CellTiter-Glo reagent was added and luminescence was measured with a Tecan Infinite M200 plate reader. Values were normalized to the untreated control sample.
7
Small Molecule Inhibitor Assay Protocol
8
Apoptosis Pathway Modulators
9
Assessing Antiproliferative Effects of 5-FU, ABT-199, and WEHI-539 on Colon Cancer Cells
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Each colon cancer cell line was treated for 72 h with 5-FU at the indicated concentrations in 96-well plates (Thermo Fisher Scientific, Inc.). Subsequently, cell proliferation was determined using the Premix WST-1 Cell Proliferation Assay (Takara Bio Inc., Kusatsu, Shiga, Japan) and an Infinite M1000 PRO microplate reader (Tecan Japan, Kawasaki, Kanagawa, Japan). The half-maximal inhibitory concentration (IC50) of 5-FU was defined as the drug concentration resulting in 50% cell survival relative to that of untreated cells. Triplicate wells were treated with various drug concentrations and average IC50 values were determined. As known antagonists of BCL2 and BCLXL in in vitro studies, ABT-199 (Selleck Chemicals) and WEHI-539 hydrochloride (MedChem Express) were used and the IC50 values of each drug were obtained, respectively.
10
Inhibition of BTK and BCL-2 in Cells
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ST2825 was purchased from MedChemExpress. The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, and B-cell lymphoma-2 (BCL-2) inhibitor ABT-199 were purchased from Selleck Chemicals. All drugs were dissolved in 100% dimethyl sulfoxide (DMSO). For all samples in all of the experiments, the final DMSO concentrations were diluted to 0.1% with cell culture media, including the vehicle controls.
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