CD8+ T lymphocytes were purified from splenocytes using a CD8+ isolation kit (Miltenyi Biotec). Purified CD8+ T lymphocytes were cultured in the presence of plate-bound anti-CD3 (10 µg/ml) with or without recombinant murine VEGF-A (50 ng/ml; Miltenyi Biotec). After 48 h of culture, cells were harvested and analyzed by cytometry or used to extract mRNA. In some experiments, anti–VEGF-R1 (20 µg/ml; R&D Systems) or anti–VEGF-R2 (10 µg/ml; clone 91202; R&D Systems) antibodies or isotype control were added to the culture medium. In some experiments, 11R-VIVIT (Merck Millipore) was added 1 h at 5 µM before the addition of VEGF-A and during the stimulation with VEGF-A.
11r vivit
Manufactured by Merck Group
Sourced in United States
11R-VIVIT is a laboratory equipment product. It is a core tool used for specific research applications. The core function of 11R-VIVIT is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Cd8 isolation kit, by Miltenyi Biotec (1 mentions)
Anti vegfr1, by R&D Systems (1 mentions)
Anti vegfr2, by R&D Systems (1 mentions)
Vegf a, by Merck Group (1 mentions)
Anti cd3, by Miltenyi Biotec (1 mentions)
Lps l2880, by Merck Group (1 mentions)
Fk506, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
U73122, by Merck Group (1 mentions)
Diazoxide, by Merck Group (1 mentions)
Glibenclamide, by Merck Group (1 mentions)
Dispase 2, by Merck Group (1 mentions)
Tak 242, by InvivoGen (1 mentions)
Fk506, by Enzo Life Sciences (1 mentions)
Pluronic, by Thermo Fisher Scientific (1 mentions)
9 phenanthrol, by Merck Group (1 mentions)
Skf96365, by Merck Group (1 mentions)
Percoll, by Merck Group (1 mentions)
4 protocols using 11r vivit
1
VEGF-A Modulation of CD8+ T Cell Activity
2
Podocyte response to LPS treatment
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The immortalized human podocytes were provided by Dr. Saleem M (University of Bristol, United Kingdom) and were cultured as previously described35 (link). The podocytes were treated as follow: 10 μg/ml, 20 μg/ml, or 50 μg/ml LPS (L-2880, Sigma Aldrich, MO, USA); 1 μM FK506; 100 nM 11R-VIVIT (480401, Merck, Germany); 1 μM cyclo-RGDfK (S7834, Selleck, USA). The detailed treatments and their combinations are indicated in the text. For transient transfection of plasmid DNA or siRNA, Lipofectamine 2000 (Invitrogen, USA) was used according to the manual instructions.
3
Modulation of Spheres Formation
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U73122, obtained from Sigma-Aldrich (St Louis, Mo, USA), 11R-VIVIT (Merck Cat. no. 480401), or vehicle (DMSO), were added at indicated concentrations during spheres formation each third day. Regarding the specificity of these compounds: U73122 is a potent, selective, and cell-permeable inhibitor of C-type phosphatidylinositol-specific phospholipases that inhibits agonist-induced phospholipase C activation (IC50 = 1-2.1 μM), but has also reported inhibiting 5-lipoxygenase. 11R-VIVIT is a competitive NFAT inhibitor peptide based on the conserved calcineurin docking site of the NFAT family. Referred to as VIVIT, this peptide interferes with calcineurin-NFAT interaction without disrupting calcineurin phosphatase activity and without affecting other signalings regulated by calcineurin, such as NFKB (9 (link)).
4
Isolation and Treatment of Microglia
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Lipopolysaccharide (LPS) [from E. coli R515 (Re), TLRgrade™] and FK506 were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Papain, dispase II, glibenclamide, 9-phenanthrol, diazoxide, SKF-96365, A23187, 1,2-bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), KN-93, and Percoll were purchased from Sigma-Aldrich (St. Louis, MO, USA). 11R-VIVIT was purchased from EMD Millipore (Billerica, MA, USA). The TLR4 signaling inhibitor, TAK-242, was purchased from Invivogen (San Diego, CA, USA). Artificial cerebrospinal fluid (aCSF) was purchased from Tocris Bioscience (Avonmouth, Bristol, UK). All culture media, sera, antibiotics, DNase I, Fluo-4-AM, and pluronic were obtained from Thermo Fisher Scientific (Waltham, MA, USA). All drugs and Fluo-4-AM were solubilized in dimethylsulfoxide (DMSO) vehicle. Papain, dispase II, and DNase I were solubilized in culture media. Sera and antibiotics were added directly to culture media.
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