Synta66

Manufactured by Merck Group

Sourced in United States

Synta66 is a high-performance laboratory equipment designed for precise analytical procedures. It offers advanced features and capabilities to support a wide range of scientific applications. The core function of Synta66 is to provide reliable and accurate data collection and analysis in a controlled laboratory environment.

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Lab products found in correlation

8 protocols using synta66

Wound Healing Assay in LN-18 Cells

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LN-18 cells were seeded into 12-well plates and fed with Dulbecco’s modified Eagle Medium supplemented with 4.5 g/l glucose and 2 mM L-glutamine (Gibco^TM, Thermo Fisher Scientific, Waltham, MA, USA), 0.1 mM non-essential amino acids (Lonza, Basel, Swiss), 1% Penicillin/Streptomycin (Gibco^TM, Thermo Fisher Scientific, Waltham, MA, USA) and 1% FBS (Sigma-Aldrich, St. Louis, MO, USA) (control) or medium supplemented with 10 µM Synta66 (Merck Millipore, Burlington, MA, USA) and grown to confluence. Scratches were created at the centre of confluent cell monolayers using a 1000 μL pipette tip. Floating cells were afterwards removed, and the resultant cell gap monitored with a Cell IQ V2 MLF Cell Imaging and Analysis System (Imagen, MA, USA) for 70 h (t1-t70).

Waldherr L., Tiffner A., Mishra D., Sallinger M., Schober R., Frischauf I., Schmidt T., Handl V., Sagmeister P., Köckinger M., Derler I., Üçal M., Bonhenry D., Patz S, & Schindl R. (2020). Blockage of Store-Operated Ca2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca2+ Selective Orai1 Pore. Cancers, 12(10), 2876.

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Temozolomide and Synta66 Storage and Handling

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Temozolomide (Merck Millipore, Burlington, MA, USA) was stored as powder at 4 °C and dissolved in the corresponding culture media immediately before use. Synta66 (Merck Millipore, Burlington, MA, USA) was stored as a 50 mM stock solution in DMSO at -80°C until dilution to a maximum DMSO concentration of 0.1% in cell culture experiments.

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Synthesis and Characterization of Novel Compounds

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All chemicals and solvents were used as supplied. SKF96365 (1), CAI (2), 2-APB (3), Pyr2 (4), Pyr3 (5), Synta-66 (6), RO2959 (7) AnCoA4 (10), leflunomide (15), Gd^3+, (18) and La³⁺ (19) were purchased from Sigma-Aldrich (UK). CM4620 (14) was purchased from MedChemExpress (Insight Biotechnology Ltd, UK). Teriflunomide (16) was purchased from APExBIO (UK). MRS1845 (11) was purchased from Santa Cruz Biotechnology (Insight Biotechnology Ltd, UK). Compounds that were not commercially available were synthesised. CM3457 (13) was synthesised by Dr Rajendra Gosain (unpublished). JPIII (17) was synthesised as previously reported [31 (link)]. The synthetic route for GSK7975A (9) and GSK5503A (8) largely followed the patent protocol and is summarised in S2 and S3 Figs, respectively [66 ]. Synthesis of the 7-azaindole series compound (12) (S4 Fig) loosely followed the patent protocol [67 ]. Detailed chemistry methods for compound syntheses can be found in S1 Appendix and NMR data for all compounds and intermediates synthesized can be found in S5–S17 Figs.

Norman K., Hemmings K.E., Shawer H., Appleby H.L., Burnett A.J., Hamzah N., Gosain R., Woodhouse E.M., Beech D.J., Foster R, & Bailey M.A. (2024). Side-by-side comparison of published small molecule inhibitors against thapsigargin-induced store-operated Ca2+ entry in HEK293 cells. PLOS ONE, 19(1), e0296065.

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Calcium Signaling Pathway Modulation in Cell Culture

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DMEM:F12 was obtained from Corning, and fetal bovine serum, horse serum, antibiotics, TNFα, human EGF and human bFGF was obtained from GIBCO. Nifedipine, methoxyverapamil hydrochloride (D-600), lanthanum chloride, Bapta-AM, caffeine, dantrolene, Stattic, Synta66, EGTA, BSA, cycloheximide and collagenase were obtained from Sigma-Merck; papain was obtained from Worthington Biochemicals. Dexamethasone, 2-APB, 5′-N-Ethylcarboxamidoadenosine (NECA), ATPγS, NNC55-0396, 78c (CD38 inhibitor), xestospongin C, FK506 and ryanodine were purchased from Tocris. Insulin and 8BrcADPr were obtained from Santa Cruz. Fura-2 AM was obtained from Invitrogen. All the lipophilic drugs were dissolved in dimethyl sulfoxide (ethanol in the case of xestospongin C). The final concentration of the solvent was ≤0.1%.

Calle-Ciborro B., Espin-Jaime T., Santos F.J., Gomez-Martin A., Jardin I., Pozo M.J., Rosado J.A., Camello P.J, & Camello-Almaraz C. (2023). Secretion of Interleukin 6 in Human Skeletal Muscle Cultures Depends on Ca2+ Signalling. Biology, 12(7), 968.

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Measuring SOCE and TRPM7 Activation

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To measure SOCE, ER Ca²⁺ store depletion was stimulated by pre-incubation with 1 μM of thapsigargin (Sigma-Aldrich, USA; #:T9033) for 20 minutes prior to the experimental protocols in Ca²⁺-free Ringer’s solution. Subsequently, under fluorescence recordings, cells were continuously perfused for 120 seconds with Ca²⁺-free Ringer’s solution, followed by a re-addition of 2 mM of extracellular Ca²⁺ in normal Ringer’s solution to measure the delta (Δ) SOCE peak. To block SOCE, cells were also preincubated with pharmacological CRAC antagonists synta-66 (5 μM) (Sigma-Aldrich, USA; #:1949) or with 3,5-bis trifluoromethyl pyrazole-BTP-2 (20 μM) (Sigma-Aldrich, USA; #:203890) for two hours [10 (link),14 (link),39 (link)]. synta-66 concentration was determined after the construction of the concentration-effect curve (Fig. S2, B). The TRPM7 agonist, naltriben (NAL, 100 μM) (Tocris Bioscience, USA; #:0892) [31 (link),35 (link)], was perfused simultaneously with the Ca²⁺ re-addition for 720 seconds. In other sets of experiments, the TRPM7 antagonist NS8593 (30 μM) (Tocris Bioscience, USA; #:4597) was simultaneously perfused during the Ca²⁺ re-addition, in the absence or presence of naltriben (100 μM), as described [35 (link)].

Souza Bomfim G.H., Costiniti V., Li Y., Idaghdour Y, & Lacruz R.S. (2020). TRPM7 activation potentiates SOCE in enamel cells but requires ORAI. Cell calcium, 87, 102187.

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Chemical Screening for Cell Viability

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Dimethylsulfoxide (DMSO), Minimum Essential Medium Eagle (EMEM, M0643), sodium bicarbonate (NaHCO3), noble agar (A5431), resazurin sodium salt (199303), Vincristine (V0400000), Lomustine (L5918), YM-58483 (Y4895), Synta66 (SML1949), GSK1016790A (G0798), HC-067047 (SML0143), AC1903 (SML2244), ML218 (SML0385), Mibefradil dihydrochloride hydrate (M5441), NNC55-0396 hydrate (N0287), Yoda1 (SML1558), Verapamil hydrochloride (V4629) and Nifedipine (N7634) were purchased from Sigma-Aldrich (Ryde, NSW, Australia). IA65 was a kind gift from Professor William Denny and Dr. Ralph Stevenson, and was synthesised as previously described [28 (link)]. CellTiter-Fluor™ Cell Viability (G6081) was obtained from Promega (Madison, WA, USA).

Sedeeq M., Maklad A., Gueven N, & Azimi I. (2020). Development of a High-throughput Agar Colony Formation Assay to Identify Drug Candidates against Medulloblastoma. Pharmaceuticals, 13(11), 368.

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T Cell Calcium Signaling Imaging

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Cells were exposed to x-ray irradiation in T₃₅ Petri dishes using an Isovolt 160 Titan E source with a voltage of 90 kV and 33.7 mA (GE Sensing & Inspection Technologies) with a dose rate of 0.055 Gy/s. Ionomycin (ab120370; Abcam), thapsigargin (Tg; T9033; Sigma-Aldrich), and the cell-permeable Ca²⁺ sensors Fluo-4 AM (F14201), Fura-2 AM (F1221), and Mag-Fluo-4 (M14206; all Thermo Fisher Scientific) were dissolved in DMSO and added to external solution immediately before experiments, with final concentrations mentioned in text. To activate human T cells, ImmunoCult Human CD3/CD28/CD2 T cell activator (short T-Ac; 10990; Stemcell Technologies) was added to the cell culture medium (25 μl per 1 ml cell suspension). The cell-permeable organelle trackers Mito-Tracker Green FM (M7514) and ER-Tracker Red (E34250), nucleus-staining Hoechst dye (H1399), and PM tracker CellMaskOrange (C10045; Thermo Fisher Scientific) were used according to the manufacturer’s recommendations, diluted in external microscopy buffer, for 30 or 10 min, respectively, at 37°C. Subsequently, cells were washed and resuspended in dye-free microscopy buffer. CRAC channels were blocked by inhibitors Synta66 (SML1949; Sigma-Aldrich) and Pyr6 (203891; Sigma-Aldrich) dissolved in DMSO and resuspended in medium or microscopic solution with final concentrations of 10 or 5 µM, respectively.

Tandl D., Sponagel T., Alansary D., Fuck S., Smit T., Hehlgans S., Jakob B., Fournier C., Niemeyer B.A., Rödel F., Roth B., Moroni A, & Thiel G. (2022). X-ray irradiation triggers immune response in human T-lymphocytes via store-operated Ca2+ entry and NFAT activation. The Journal of General Physiology, 154(5), e202112865.

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Dissecting T-cell Activation Pathways

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Ionomycin stimulation was performed by perfusing Ionomycin (Invivogen) at 10 μM final concentration. Antibody stimulations were performed by pairing CD8 + T cells with anti-CD3/CD28-coated microbeads (Thermofischer Scientific). For SOC channels activity experiments, thapsigargin (focus biomolecules) was added at 2 μM final concentration, and then a 2 mM Ca 2+ solution was subsequently injected 6 minutes later. Calcium channel inhibitors Synta66 (Sigma Aldrich) and BTP2 (Abcam) were added at a final concentration of 10 μM to assess the implication of SOC calcium channels in the thapsigargininduced calcium entry.
For the cell pairing experiments, first, allogenic healthy CD8 + T lymphocytes were activated by anti-CD3/CD28 beads at a 1 : 1 ratio for 3 days and cultured 7 days with 30 U ml -1 of human recombinant IL-2. Activated allogenic healthy CD8 + T lymphocytes were subsequently pulsed with or without 1 μg ml -1 of a cocktail of superantigens (SEA and Tsst-1; Sigma-Aldrich) for 30 min at 37 °C and AML blast cell was stained with membrane dye DiI (Thermo Fischer Scientific) before cell-cell pairing experiments.

Shaik F.A., Lewuillon C., Guillemette A., Ahmadian B., Brinster C., Quesnel B., Collard D., Touil Y., Lemonnier L, & Tarhan M.C. (2022). Pairing cells of different sizes in a microfluidic device for immunological synapse monitoring. Lab on a chip, 22(5).

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