Primary rat astrocytes were grown to 80% confluence in 15 cm dishes before being incubated in the presence or absence of EVs at a concentration of 1 × 109 particles/mL for 72 hours. EV-treated astrocytes were re-plated at 1 × 106 cells/well into 6-well dishes precoated with 0.2% gelatin, which was subsequently crosslinked with 1% glutaraldehyde for 30 min, quenched in 1 M glycine for 20 min. Astrocytes were allowed to deposit ECM for 6 days in medium supplemented with 50 µg/mL ascorbic acid in the presence or absence of the DGK-inhibitor R59022 (10 µM) (Sigma) or hyaluronidase (Hase) (50 µg/mL; Type I-S, Sigma H3506) where indicated. ECM was then decellularized by incubation with PBS containing 20 mM NH4OH and 0.5% Triton X-100.
Type 1 s
Manufactured by Merck Group
Sourced in United States, United Kingdom
The Type I-S is a piece of laboratory equipment designed for the separation and purification of biological samples. It utilizes a centrifugal force to separate components based on their density and size. The device is suitable for a range of applications in research and diagnostic settings.
Automatically generated - may contain errors
Lab products found in correlation
R59022, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Krebs ringer bicarbonate buffer, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
Ethanol, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Thiazolyl blue tetrazolium bromide, by Merck Group (1 mentions)
Potassium dihydrogen phosphate, by Thermo Fisher Scientific (1 mentions)
Sodium phosphate dibasic, by Thermo Fisher Scientific (1 mentions)
6 protocols using type 1 s
1
Astrocyte-Derived ECM Modulation
2
Modulating Synovial Fluid Viscosity
3
Bovine Testes Hyaluronidase Preparation
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Type I-S (lyophilized powder, approximately 400–1000 units/mg solid, Sigma-Aldrich, St. Louis, MO, USA) hyaluronidase from bovine testes was used, with an average activity of 748 units/mg. A total of 13.36 mg of hyaluronidase powder was mixed with 20 mL of purified water to obtain a concentration of 10,000 IU/mL. After dissolving the powder completely, a 0.22 μm syringe filter was used to filter the solution to remove any existing particles of hyaluronidase powder.
4
Cell Culture and Enzymatic Dissociation
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Rat 9L gliosarcoma cells were a gift from Dr Hrvoje Miletic (University of Bergen, Norway) and human M21 Melanoma cells were provided by Dr David Cheresh (University of California, La Jolla). Human LN229 and SNB19 glioblastoma cells were provided by Dr Nelofer Syed (Imperial College London, UK), LNCaP prostate cancer cell line was a gift from Dr Paul Mintz (Imperial College London) and the mouse C2C12 myoblast cell line was provided by Dr Francesco Muntoni (University College London, UK). The human breast cancer MCF-7 and glioblastoma U87 cell lines were from Cancer Research UK. Cells were sustained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) supplemented with 10 % Fetal Bovine Serum (FBS, Sigma), L-Glutamine (2 mM, Sigma), Penicillin (100 units/ml, Sigma) and Streptomycin (100 μg/ml, Sigma). The C2C12 cells were grown in 20 % FBS. Cells were maintained at 37 °C in a humidified atmosphere supplemented with 5 % CO2. Collagenase from Clostridium histolyticum (Type I, ≥125 CDU/mg, Sigma) and hyaluronidase from bovine testes (Type I-S, 400-1000 units/mg, Sigma) were solubilized in phosphate buffered saline (1x PBS, Sigma).
5
In Vitro Cytotoxicity Evaluation
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Metolose (MET) was obtained from Shin Etsu (Stevenage, Hertfordshire, UK), polyethylene glycol (PEG 400), L-arginine (L-arg), gelatine and mucin from bovine submaxillary gland, Type I-S, Krebs-Ringer bicarbonate buffer, thiazolyl blue tetrazolium bromide, MTT reagent [(3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Gillingham, UK). Omeprazole (OME) was obtained from TCI (Tokyo, Japan). Ethanol, potassium di-hydrogen phosphate, sodium hydroxide, sodium chloride, sodium phosphate di-basic were all obtained from Fisher Scientific (UK). Dulbecco's Modified Eagles Medium (DMEM), foetal bovine serum (FBS), penicillin, streptomycin and glutamine were all obtained from Gibco (Paisley, UK).
6
Astrocyte-Derived Extracellular Matrix
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Primary rat astrocytes were grown to 80% confluence in 15cm dishes before being incubated in the presence or absence of EVs at a concentration of 1 X 10 9 particles/ml for 72 hr. EVtreated astrocytes were re-plated at 1 X 10 6 cells/well into 6-well dishes pre-coated with 0.2% gelatin, which was subsequently crosslinked with 1% glutaraldehyde for 30 min, quenched in 1M glycine for 20min. Astrocytes were allowed to deposit ECM for 6 days in medium supplemented with 50 μg/mL ascorbic acid in the presence or absence of the DGK-inhibitor R59022 (10μM) (Sigma) or hyaluronidase (Hase) (50 µg/mL; Type I-S, Sigma H3506) where indicated. ECM was then de-cellularised by incubation with PBS containing 20mM NH 4 OH and 0.5% Triton X-100.
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