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Fk506

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FK506 is a macrolide compound that functions as an immunosuppressant. It inhibits T-cell activation and proliferation by binding to the immunophilin FKBP12, which in turn inhibits the phosphatase activity of calcineurin.

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Lab products found in correlation

Co2 incubator, by Thermo Fisher Scientific (3 mentions) Porcine skin gelatin, by Merck Group (2 mentions) Rat tail collagen 1, by Thermo Fisher Scientific (2 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Mcdb131 media, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Thapsigargin, by Bio-Techne (1 mentions) Nimodipine, by Bio-Techne (1 mentions) D serine, by Bio-Techne (1 mentions) Am251, by Abcam (1 mentions) Bapta, by Bio-Techne (1 mentions) Gdp β s, by Bio-Techne (1 mentions) Ttx citrate, by Abcam (1 mentions) Ly341495, by Abcam (1 mentions) Fluoroacetate, by Merck Group (1 mentions) D ap5, by Abcam (1 mentions) Mk 801 maleate, by Abcam (1 mentions) Rat igg2a isotype control, by BioXCell (1 mentions) Ly294002, by LC Laboratories (1 mentions)

11 protocols using fk506

1

Mesangial Cell Culture and RNA Extraction

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Primary human mesangial cells (Lonza, Clonetics, CC-2559) were cultured in MCDB-131 media (Gibco-invitrogen), supplemented with 10% FBS, 100U penicillin/ml 100ug/ml streptomycin and 2mmol L-Glutamine (all supplied from Gibco-Invitrogen) at 37°C in humid atmosphere with 5%CO2 in air. Cells were seeded onto six-well cell culture plates (Greiner Bio one) and were used for experiments at the fifth passage. Confluent cells were cultured in serum free media and FK 506 (Abcam) dissolved in DMSO (Sigma) in a final concentration of 1 μM for 24h. The control group was treated in serum free media and DMSO only and a total of four independent experiments were conducted. Trizol® and chloroform reagent was used to extract RNA (Invitrogen). The RNA pellet was dissolved in 10 μl sterile water and stored at –80°C.
Aschauer C., Perco P., Heinzel A., Sunzenauer J, & Oberbauer R. (2017). Positioning of Tacrolimus for the Treatment of Diabetic Nephropathy Based on Computational Network Analysis. PLoS ONE, 12(1), e0169518.
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2

Pharmacological Agents for Synaptic Plasticity

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Pharmacological agents were purchased from: Sigma-Aldrich: Fluoroacetate, thapsigargin, BAPTA, bicuculline methbromide, Tricine, Zinc chloride, d-serine, and GDPβS; Tocris Bioscience: nimodipine, (+)-MK-801 maleate, d-AP5, NBQX, TTX citrate, PPDA, Ro 25-6981 maleate, MCPG, MPEP, LY341495, AM251, 2-AG, and FK506; and Abcam: UBP-141. Salts used for internal and external solutions were purchased from Sigma-Aldrich. Compounds were dissolved in H2O or Ringer solution with the exception of thapsigargin, nimodipine, PPDA, FK506, THL, AM251, and 2-AG, which were dissolved in DMSO. Vehicle (DMSO) at the concentrations used did not affect baseline EPSP amplitudes and had no other detectable effects on the neurons. When investigating the effect of pharmacological agents on plasticity, all drugs were included in the superfusion fluid or patch pipette from the start of the experiment until completion (from 0 to 50 min in a standard plasticity experiment), except for Fluoroacetate, which was applied from 60 min before the start of recording. When determining the effect of a pharmacological agent on baseline condition, a stable baseline of at least 10 min was first recorded and then the drug was bath applied by switching to a different perfusion line.
Andrade-Talavera Y., Duque-Feria P., Paulsen O, & Rodríguez-Moreno A. (2016). Presynaptic Spike Timing-Dependent Long-Term Depression in the Mouse Hippocampus. Cerebral Cortex (New York, NY), 26(8), 3637-3654.
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3

NOD Mouse Immunomodulation Protocols

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NOD/LtJ and NOD.Cg-Tg(TcraBDC2.5)1Doi Tg(TcrbBDC2.5)2Doi/DoiJ (NOD.BDC) mice were bred and maintained in specific pathogen-free facilities at the University of North Carolina at Chapel Hill (UNC-CH). All animal experiments were approved by the UNC-CH Institutional Animal Care and Use Committee.
12 wk-old nondiabetic NOD female mice were used for all experiments. Animals received a single i.p. injection of 600 μg of the αCD4 clone YTS177.9 or 2A3, a rat IgG2a isotype control (BioXCell). In some experiments NOD mice were injected i.p. with 75 mg/kg of Ly294002 (LC Laboratories) 6 h prior to Ab treatment, and then daily for 3 d. Alternatively, NOD mice received 3 mg/kg of NSC237666 (Santa Cruz Biotechnology) i.p. 2 h prior to Ab treatment, and then every 12 h for 3 d. For calcineurin inhibition, NOD mice were injected i.p. with 1 mg/kg of FK506 (Abcam) 6 h prior to Ab treatment, and then daily for 5 d.
Morillon YM I.I., Lessey-Morillon E.C., Clark M., Zhang R., Wang B., Burridge K, & Tisch R. (2016). Antibody binding to CD4 induces Rac GTPase activation and alters T cell migration. Journal of immunology (Baltimore, Md. : 1950), 197(9), 3504-3511.
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4

Isolation and Activation of Rat CD4+ T Cells

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Unless otherwise stated all cell culture materials and chemical reagents were purchased from Sigma Aldrich (Gillingham, UK) or Thermo Fisher Scientific (Loughborough, UK). Alginic acid sodium salt powder was purchased from Sigma Aldrich (Gillingham, UK). FK506 was obtained from Abcam (Cambridge, MA, USA). MagCellect Rat CD4 + T cell isolation kits were sourced from R&D systems (Minneapolis, MN, USA). Cell activation cocktail was provided by Biolegend (San Diego, CA, USA). A FK506 enzyme linked immunosorbent assay (ELISA) kit was purchased from Abnova (Taipei City, Taiwan). Running buffer and calibration beads for flow cytometry were obtained from Miltenyi Biotec (Gladbach, Germany).
Collection and use of tissue from animals were conducted in accordance with the UK Animals (Scientific Procedures) Act (1986) and the European Communities Council Directives (86/609/EEC) and approved by the UCL Animal Welfare and Ethical Review Body.
Eleftheriadou D., Evans R.E., Atkinson E., Abdalla A., Gavins F.K., Boyd A.S., Williams G.R., Knowles J.C., Roberton V.H, & Phillips J.B. (2022). An alginate-based encapsulation system for delivery of therapeutic cells to the CNS. RSC Advances, 12(7), 4005-4015.
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5

Culturing HEK 293 and ES Cells

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HEK 293 cells (ATCC) were cultured using standard conditions in media containing: DMEM (Life Technologies), 10% FBS (Applied StemCell), and Penicillin-Streptomycin (Life Technologies).
ES cells were cultured using standard conditions in media containing: DMEM (Life Technologies), 7.5% ES-sure FBS (Applied StemCell), 7.5% KnockOut SR (Life Technologies) Penicillin-Streptomycin (Life Technologies), Glutamax (Life Technologies), HEPES buffer (Life Technologies), 2-mercaptoethanol (Life Technologies), and MEM-NEA (Life Technologies). LIF was replaced daily and ES cells were passaged every 48 h.
For long recruitment experiments (>1 h), cells were treated with 3 nM RAP (Selleckchem) and/or 3 nM FK506 (Abcam) added directly to culture dish and changed daily. For short recruitment experiments (≤1 h), cells were treated with 30 nM RAP and/or FK506 in suspension.
Braun S.M., Kirkland J.G., Chory E.J., Husmann D., Calarco J.P, & Crabtree G.R. (2017). Rapid and reversible epigenome editing by endogenous chromatin regulators. Nature Communications, 8, 560.
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6

Biomimetic Sponges for Tissue Repair

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Biomimetic sponges were fabricated as reported previously [20 ]. Briefly, a porcine skin gelatin (3 wt %, Sigma‐Aldrich) was combined with EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (20 mM, Thermo Fisher Scientific) and rat tail collagen I (Gibco, 3 mg/mL) at a 70:30 gelatin: collagen ratio. Laminin (LM)-111 (50 μg/mL, Trevigen) and FK506 (25 μM, Abcam) were added to the solution and aliquoted into 48-well plates at 700 μL/well. The solution was allowed to gel and was subjected to slow freezing for 48 hours before lyophilization. Prior to implantation, the sponges were disinfected with ethanol for five minutes, rinsed twice with sterile phosphate-buffered saline (PBS) and incubated in sterile PBS overnight.
Dunn A., Haas G., Madsen J., Ziemkiewicz N., Au J., Johnson D., West C., Chauvin H., Gagyi S.M, & Garg K. (2021). BIOMIMETIC SPONGES IMPROVE FUNCTIONAL MUSCLE RECOVERY FOLLOWING COMPOSITE TRAUMA. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 40(5), 1039-1052.
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7

Combinatorial Therapy for Neuroprotection

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FK506 and GPI1046 were prepared and applied as described previously13 (link). Briefly, mice were treated orally with 0.2 mg/kg body weight per day FK506 (Abcam Biochemicals, Cambridge, UK) or subcutaneously with 10 mg/kg body weight per day GPI1046 (Santa Cruz Biotechnology, Dallas, USA) with the combination of LB100 once daily starting one day prior of surgery. LB100 (Selleck Chemicals, USA) was prepared by dissolving in sterile PBS at a concentration of 50 mM stock solution. One day before surgery, mice were injected subcutaneously with 2 mg/kg body weight per day LB100. Okadaic acid (Cell signaling technology, USA) 1 mM stock solutions were prepared by dissolving in DMSO. Tautomycetin (Tocris, USA) was supplied as pre-dissolved in DMSO at a concentration of 10 mg/ml.
Nyamsuren G., Rapp G., Dihazi H., Zeisberg E.M., Tampe D., Tampe B, & Zeisberg M. (2021). PP2A phosphatase inhibition is anti-fibrotic through Ser77 phosphorylation-mediated ARNT/ARNT homodimer formation. Scientific Reports, 11, 24075.
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8

Fabrication of Gelatin-Collagen Sponges

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A 3 wt% porcine skin gelatin (Sigma–Aldrich) solution was prepared in deionized (DI) water and heated to 60°C. After the gelatin had completely dissolved, the solution was allowed to cool to 50°C. The cooled gelatin solution was combined with 20 mM of EDC. Rat tail collagen I (Gibco, 3 mg/ml) was mixed with the gelatin solution at a ratio of 70:30 by volume. LM-111 (Trevigen) were added to the solution at final concentrations of 50 μg/ml. FK-506 (Abcam) was added at a final concentration of 25 μM, respectively. The solution was then aliquoted into a 48-well plate at 700 μl/well. The well plate was placed in a 100% methanol bath, allowing the sponges to gel at 4°C for 30 min, followed by overnight freezing at −8°C. The well plate was then removed from the bath and moved to a − 80°C freezer for 48 hr and subsequently lyophilized for at least 12 hr. Prior to in vitro culture or surgical implantation, the sponges were disinfected using ethanol and rinsed twice in sterile 1× phosphate-buffered saline (PBS).
Haas G., Dunn A., Madsen J., Genovese P., Chauvin H., Au J., Ziemkiewicz N., Johnson D., Paoli A., Lin A., Pullen N, & Garg K. (2021). Biomimetic sponges improve muscle structure and function following volumetric muscle loss. Journal of biomedical materials research. Part A, 109(11), 2280-2293.
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9

Pinostrobin Extraction and Purification

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Pinostrobin was extracted and purified from the rhizomes of B. pandurata as described in our previous report (Wangkangwan et al. 2009 ). GSK3 inhibitor I (Calbiochem, Darmstadt, Germany), FK506 (Abcam, Cambridge, UK) and radicicol (Sigma-Aldrich, St. Louis, MO, USA) were obtained commercially.
Sopanaporn J., Suksawatamnuay S., Sardikin A., Lengwittaya R., Chavasiri W., Miyakawa T, & Yompakdee C. (2020). Pinostrobin suppresses the Ca2+-signal-dependent growth arrest in yeast by inhibiting the Swe1-mediated G2 cell-cycle regulation. FEMS yeast research, 20(4).
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10

Culturing Human Cell Lines for Research

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Human normal esophageal epithelial cell line HEEC was cultured in DMEM (High Glucose) with 10% fetal bovine serum (FBS). Human esophageal cancer cell lines (EC109, KYSE-150, TE-1 and TE-10), human gastric cancer cell line HGC-27, human hepatocarcinoma cell line HepG2, and human breast cancer cell line MDA-MB-231 were cultured in DMEM with 10% FBS. Human keratinocyte cell line HaCaT and human lung cancer cell line A549 were cultured in RPMI 1640 medium with 10% FBS. The medium and FBS used in the present study were purchased from Gibco (U.S.A.). All the cell lines were cultured at 37°C in the CO2 incubator (Thermo Fisher Scientific, U.S.A.). RQ-00203078 (10 nM, APExBIO, U.S.A.), WS 12 (500 nM, Abcam, U.S.A.), FK506 (10 μM, Abcam, U.S.A.), purified anti-human PD-L1 neutralizing antibody (5 μg/ml, BioLegend, U.S.A.), and purified human IgG2 isotype control recombinant antibody (5 μg/ml, BioLegend, U.S.A.) were used to treat EC109 cells in the following experiments.
Lan X., Zhao J., Song C., Yuan Q, & Liu X. (2019). TRPM8 facilitates proliferation and immune evasion of esophageal cancer cells. Bioscience Reports, 39(10), BSR20191878.
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