After reaching 90–95% confluence, astrocyte monolayers were washed with PBS, incubated with serum-free Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) for 24 h to allow cells to reach a non-dividing G0-phase in the cell cycle (14 (link)), then treated with or without 20 µM FK506 (Sigma-Aldrich) in serum-free DMEM for 24 h. The conditioned media (CM) of the control group (C-CM) and FK506-treated group (FK506-CM) were collected, centrifuged at 7,500 × g for 20 min by an Amicon Ultra-4 3K centrifugal filter device (Merck Millipore) to remove residual FK506, then diluted to the initial volume with neurobasal medium (Gibco; Thermo Fisher Scientific, Inc., Grand Island, NY, USA). Subsequently, the conditioned media were stored at −80°C and used within one week.
Fk506
Manufactured by Merck Group
Sourced in United States, Germany, Macao
FK506 is a laboratory product used in research applications. It functions as an immunosuppressant agent.
Automatically generated - may contain errors
Lab products found in correlation
Cyclosporin a, by Merck Group (7 mentions)
Ionomycin, by Merck Group (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Rapamycin, by Merck Group (6 mentions)
Thapsigargin, by Merck Group (4 mentions)
Neurobasal medium, by Thermo Fisher Scientific (4 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Cyclosporine a, by Merck Group (3 mentions)
Nim811, by Novartis (3 mentions)
Fetal bovine serum (fbs), by PAN Biotech (3 mentions)
U0126, by Merck Group (3 mentions)
Amphotericin b, by Merck Group (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Gsk2606414, by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Mk 2206, by Selleck Chemicals (2 mentions)
Bapta am, by Thermo Fisher Scientific (2 mentions)
Kn 62, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Trolox, by Merck Group (2 mentions)
98 protocols using fk506
1
Astrocyte Conditioned Media Preparation
2
Investigating FXR Role in Tacrolimus-Induced Diabetes
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To test if the FXR is involved in tacrolimus-induced diabetes mellitus, we purchased human renal cortex proximal tubule epithelial (HK-2) cell lines (Stem Cell Bank of the Chinese Academy of Sciences, No. CRL-2190TM) and divided them into three groups: (1) the control group: HK-2 cells were cultured routinely with dimethyl sulphoxide (DMSO) solution (Sigma-Aldrich, USA, No. 34869-100ML); (2) the FK506 group: HK-2 cells were treated with 15 µmol/L FK506 (Sigma-Aldrich, USA, No. F4679) for 24, 48 and 72 h; and (3) the GW4064 group: HK-2 cells were treated with 4 μmol/L GW4064 for 24, 48 and 72 h. We collected all cell samples for mRNA and protein detection.
3
T Cell Activation Regulation by Pharmacological Modulators
4
Evaluating Cell Death Mechanisms
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DAPI (4 ,6-diamidino-2-phenylindole, Cat No. D9592), propidium iodide (PI, Cat No. P4170), staurosporine (Cat No. S6942) and FK506 (tacrolimus, Cat No. F4679) were purchased from Sigma-Aldrich (Bornem, Belgium). FTI-277 was a kind gift from Janssen Pharmaceutica (Beerse, Belgium). 1 mg/ml stock solutions of PI (40×) and DAPI (10,000×) were made in H 2 O. staurosporine and FTI-277 were dissolved in DMSO (Sigma-Aldrich, Cat No. 276855) in a stock solution of 1 mM. FK506 was dissolved in DMSO in a stock solution of 10 mM (1000×). For Western blot the antihuman anti-␣-SYN antibody 15G7 (1:100, Enzo Life Sciences, Cat No. ALX-804-258) was used and a mouse monoclonal anti-␣tubulin antibody (1:50,000, Sigma, Cat No. T5168) was used as internal loading control. The rabbit polyclonal antibody against cleaved caspase-3 was purchased from Cell Signaling Technology (Danvers, MA, USA Cat No. 9661s), and used at 1:500 and 1:1000 for immunocytochemistry (ICC) and western blotting, respectively. The cytotoxicity detection kit was purchased from Roche (Cat No. 11644793001 ) and the lactate dehydrogenase activity was determined according to the manufacturer's instructions.
5
Synthetic Aβ Oligomers: A Tractable Tool for Alzheimer's Research
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Full-length, synthetic Aβ 1–42 peptides (AβOs; American Peptide, Sunnyvale, CA) were prepared exactly according to the method of Lambert et al. (1998) (link) and applied to 11–13 DIV cells at a final concentration of 500 nM for 18 h. We use synthetic AβOs in our studies for the following reasons. First, they mimic the toxic properties of natural oligomers (brain or cell derived) as described previously (Jin et al., 2011 (link); Welzel et al., 2014 (link)). Second, unlike natural oligomers, synthetic AβOs can be detected by immunocytochemistry. Confirmation of AβO binding is crucial in our experiments because it varies considerably between neurons. Although they may not be identical to natural oligomers, synthetic AβOs are a tractable tool for investigating mechanisms of AD pathogenesis (Ferreira and Klein, 2011 (link)). After AβO or vehicle exposure, cells were incubated with 1 μM FK506 (Sigma-Aldrich, St. Louis, MO) or equivalent volumes of vehicle (ethanol) for 1–3 h before imaging of transport. For all VGCC inhibition experiments, cells were incubated with 100 μM conotoxin GVIA (Alomone Labs, Jerusalem, Israel), 50 μM agatoxin IVA (Alomone Labs), or 10 μM nimodipine (Tocris Bioscience, Bristol, United Kingdom) for 30 min before AβO treatments. For all RyR inhibition experiments, cells were incubated with 0.5 μM dantrolene (Sigma-Aldrich) for 1 h before AβO treatment.
6
Pharmacological Inhibitors for Cell Signaling
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SB202190, GSK2606414, thapsigargin (Tg), FK506, chloroquine (CQ), and cyclosporin A (CsA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Torin 1 was from (Tocris-Biotechne, Minneapolis, MN, USA).
7
OT-I Rag−/− Splenocyte Stimulation
8
Chemical Inhibitors of Cell Signaling Pathways
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BEZ235, CAL-101, GSK650394, Roscovitine, Salubrinal, AZD8055, MK-2206 and PF4708671 were from Selleck Chemicals LLC (Houston, TX, USA). CN585 (6-(3,4-Dichlorophenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine) was from Merck Millipore (Merck, Damstadt, Germany). Ionomycin, PMA, CsA, FK-506 and BV-02 were from Sigma-Aldrich.
9
Zebrafish Tremblor Mutant Cardiac Evaluation
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Zebrafish tremblor tc318 heterozygotes were bred in the Tg(myl7:EGFP) background and raised as previously described (Westerfield, 2000 ). Embryos were raised at 28.5°C and staged as previously described (Kimmel et al., 1995 (link)). For Cn or proteasome inhibition, embryos were treated with 10 μM FK506 (Sigma-Aldrich, St. Louis, MO) or 50 μM MG132 (Sigma-Aldrich) at 24 hpf. The morpholino-modified antisense oligonucleotides targeting the translation initiation sites of murf1a and murf1b (Table 1 , Gene Tools) were microinjected at the 1- to 2 cell stage (8 ng each). This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of the University of California, Los Angeles. The protocol was approved by the Chancellor's Animal Research Committee of the University of California, Los Angeles.
10
Osteogenic Differentiation of Mesenchymal Stem Cells
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Cells at passage 3 with 80% confluence were trypsinized using TrypLE Express and seeded into six plates at 1×104 cells/cm2 and incubated with Mesenchymal Stem Cell Growth medium at 37°C for 24 h. Following incubation, the growth medium was replaced with 2 ml osteo-induction medium was prepared as previously described (12 (link)) containing 10 mM Na β-glycerophosphate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 0.25 mM l-ascorbic acid (MP Biomedicals, LLC, Santa Ana, CA, USA) and 0, 5, 50, 500 or 5,000 nM FK506 (Sigma-Aldrich; Merck KGaA). The osteo-induction medium was replaced every other day; cells were collected at 3, 7 and 14 days for RT-qPCR analysis, and phenotype staining was done at 7 or 14 days. All biochemical assays were carried out at least three times.
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