Adult wild type zebrafish (Danio rerio) were originally obtained from Carolina Biological and have been maintained at Brown University as a genetically diverse outbred strain. For the analysis of structural brain defects, we used the Tg(elavl3:EGFP) line, which expresses the enhanced green fluorescent protein under control of a ubiquitous neuronal promotor [26 (link)]. Zebrafish embryos were collected within one hour after spawning and raised at 28.5°C in egg water, containing 60 mg/L sea salt (Instant Ocean) in deionized water and 0.25 mg/L methylene blue as a fungal inhibitor. Cyclosporine (cyclosporin A, Enzo Life Sciences) and FK506 (tacrolimus, Enzo Life Sciences) were diluted in egg water from 1000x stocks dissolved in dimethylsulfoxide (DMSO). The corresponding DMSO concentration (0.1% DMSO) was used as a control. Embryos were exposed from 2-26 hours post-fertilization (hpf), 26-50 hpf, or 50-74 hpf, washed four times in egg water, and grown in egg water for up to 5 days post-fertilization (dpf). The developing zebrafish are referred to as ‘embryos’ from 0-3 dpf and as ‘larvae’ afterwards [27 ].
Tacrolimus
Manufactured by Enzo Life Sciences
Tacrolimus is a potent immunosuppressant drug used in the prevention of organ rejection following transplantation. It is a macrolide compound produced by the bacterium Streptomyces tsukubaensis.
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Lab products found in correlation
3 protocols using tacrolimus
1
Structural Brain Defects in Zebrafish
2
Immune Suppressive Agent Response in THP-1 Cells
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THP-1 cells cultured in RPMI 10% FCS were plated at a density of 200 000 cells/well in 96-well plates and pretreated for 4 h at 378C with serial dilutions of the immune suppressive agents tacrolimus, mycophenolate mofetil and rapamycin (all Enzo Life Sciences, Farmingdale, NY). The dose range for each drug was defined to ensure comparable metabolic activity between treated and untreated cells, as per MTT assay (data not shown; Roche, Indianapolis, IN). IFN-g was added (final concentration 1 mg/mL; Peprotech, Rocky Hill, NJ) and cells were incubated for an additional 6 h before RNA extraction. Cells were lysed in plate and automated RNA extraction was performed using an RNeasy Micro kit (Qiagen).
3
Monoclonal Antibody-Induced Endothelial Stimulation
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Mouse IgG2a anti-human monoclonal HLA class I antibody clone W6/32 (Sigma-Aldrich, St Louis, MO), mouse IgG2a anti-human monoclonal HLA-DR, DP, DQ clone Tü39 (BD Biosciences, San Diego, CA), and clone WR18 (ThermoFisher Scientific, Rockford, IL) at 1 or 2 μg/mL were used to stimulate endothelial cells for 10 minutes or 1 hour. The concentrations of anti-HLA antibodies used are the expected physiological concentrations in human blood, as measured after the purification of DSAs from patients using specific immunoaffinity columns. 17 Stimulation with histamine (Prepotech, Rocky Hill, NJ) at 1.0 mmol/L for 10 minutes or thrombin at 1 U/mL (ThermoFisher Scientific) for 1 hour was used as a positive control. Tacrolimus was obtained from Enzo Life Sciences Inc. (Farmingdale, NY). Assessment of cell viability and the methods used to test cyclosporine, prednisone, and mycophenolic acid are described as Supplemental Material.
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