Gum Arabic (GA) used in this study was purchased from the ENNASR company (Mw: 2.5 × 106 g. mol−1, Khartoum, Sudan). Sodium alginate (ALG: molecular weight = 4.8 × 106 ± 1.8 × 105 Da, nominal viscosity = 300 mPa.s) was bought from Sigma Aldrich (Dteroit, MI, USA). Curcumin (purity, >98%, Mw = 368.36 g/moL, C12H20O6) was bought from Biolution Resources company (Serdang, Selangor, Malaysia). Phosphate buffer solution (PBS) was purchased from R&M (Pekin, China). Fetal Bovine Serum (FBS), Trypsine-EDTA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), Trolox, and Dulbecco’s modified Eagle’s medium (DMEM) high glucose were bought from Sigma Aldrich (St. Loius, MO, USA).
Trypsine edta
Manufactured by Merck Group
Sourced in United States
Trypsin-EDTA is a laboratory reagent used for cell detachment and dissociation. It contains trypsin, a proteolytic enzyme, and EDTA, a chelating agent. Trypsin-EDTA is commonly used to break down the extracellular matrix and cell-to-cell junctions, allowing adherent cells to be harvested for subculturing or other downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Sodium alginate alg, by Merck Group (1 mentions)
1 1 diphenyl 2 picrylhydrazyl dpph, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Trolox, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem high glucose, by Merck Group (1 mentions)
Ksom aa, by Merck Group (1 mentions)
M2 medium, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dmem high glucose, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Mccoy s 5a, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Nadph, by Merck Group (1 mentions)
Ammonium formate, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Merck Group (1 mentions)
Soy l α phosphatidylcholine, by Merck Group (1 mentions)
2 hydroxypropyl beta cyclodextrin mw 1460, by Merck Group (1 mentions)
6 protocols using trypsine edta
1
Curcumin-loaded Alginate-Gum Arabic Hydrogels
2
Single-cell isolation from mouse oocytes
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Oocyte and embryo collection, in vitro culture and single cell isolation. F1 female mice (C57BL/6 x DBA/2) were superovulated and in the case of embryo collection, mated to C57BL6 males. MII oocytes were collected 14 hours post hCG injection in M2 medium (Sigma, M7167). Zygotes were collected 14–16 hours post hCG collection in M2 medium. Cumulus cells were removed by incubation for 5 min in 1 mg/ml Hyaluronidase-containing M2. Embryos were cultured in KSOMaa (Milipore, MR-106-D) in a low oxygen air chamber. Single blastomeres were isolated by first removing the Zona pellucida with Proteinase (Sigma, P5147-1 G, 5 mg/ml in M2 medium), followed by separating the cells with Trypsine (Trypsine-EDTA, T4049 Sigma). Single cells were picked with the help of a mouth pipette and a finely pulled glass capillary. All experiments were performed in accordance with the Swiss Animal Protection laws and institutional guidelines and were approved by Cantonal Veterinary Office (Basel-City, Switzerland).
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3
Patch-Clamp Recordings of Transfected HEK-293 Cells
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For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA). Cells were maintained in 25 cm2 flasks or 6‐well plates at 37°C (5% CO2). Before transfection, the cells were washed with phosphate‐buffered saline (PBS) for 1 min then treated with 0.25% trypsine‐EDTA (Sigma Aldrich, St. Louis, MO, USA). The cells were then seeded onto 35 mm petri dishes (Carl Roth, Karlsruhe, Germany) containing 22 mm glass coverslips (Carl Roth, Karlsruhe, Germany). After 24–48 h of incubation, we transfected these cells using a mixture of 2.5 μg of hERG plasmid DNA and 1.5 μg of enhanced green fluorescent protein N1 (EGFP‐N1) and Lipofectamine 2000 (Thermofisher Scientific, Waltham, MA, USA). We used the cells for experiments 24–96 h after transfection.
4
Synthesis and Characterization of Unsymmetrical Bisacridines
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Unsymmetrical bisacridines, UAs, C-2028, C-2045 and C-2053 were synthesized in the Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, as described previously [29 ,30 ]. The following chemicals were obtained from Merck (Darmstadt, Germany): methanol (gradient grade for liquid chromatography), HCl, HEPES, alamethicin, NADPH and UDPGA. Ammonium formate was from Fisher Scientific (Pittsburgh, PA, USA). All other chemicals and solvents: H3BO3 (p.a. POCH, Poland), NaOH (p.a., POCH, Poland), NaCl (p.a., POCH, Poland) were of the highest purity available and were used as provided by manufacturers. Cell culture media McCoy’s 5A, antibiotics and trypsine-EDTA were obtained from Sigma Aldrich/Merck (Darmstadt, Germany), while fetal bovine serum (FBS) from Biowest (Nuaille, France).
5
Fenretinide Lipid Formulation Optimization
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N-4-hydroxyphenyl-retinamide (fenretinide, 4-HPR) was purchased from Olon Spa (Milan, Italy); spermidine, soy L-α-phosphatidylcholine, glyceryl tributyrate, 2-hydroxypropyl beta cyclodextrin (Mw 1460), and KOH from Sigma-Aldrich (Milan, Italy); ethanol absolute anhydrous from Carlo Erba Reagents (Milan, Italy). DMEM medium, dichlorofluorescein diacetate (DCHFDA), Hoechst 33342, glutamine, trypsine/EDTA solutions, and Human Serum were from Sigma-Aldrich.
6
Cytotoxic CD8+ T Cell Assay
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Using the CD8α+ T-Cell-Isolation-Kit (Miltenyi Biotec), the CD8+ T-cell fraction was enriched from an OT-I mouse-derived splenic single-cell suspension. Next, 1e6 OT-I cells were resuspended in 1 ml RPMI-1640+ with 50 μmol/L β-mercaptoethanol and stimulated for two days with 20 μl Mouse T-Activator CD3/CD28 Dynabeads (Gibco). To evaluate target cell-specific killing, 6k LLC-eGFP/OVA cells were mixed with 6k LLC-Kat cells, 11k monocytes and 3k stimulated OT-I cells to obtain a target:effector ratio of 2:1 in 250 µl DMEM+. The added monocytes were either in vitro differentiated from bone marrow cells or isolated and MACS enriched using the Monocyte Isolation Kit and CD45 microbeads (Miltenyi Biotec, #130-100-629 and 130-052-301 resp.). While supernatants were collected 72hrs later for TNF-α and IFN-γ ELISA, cells were detached with 50 µl Trypsine-EDTA (Sigma-Aldrich) for 5 min at 37°C. LLC target cell-specific OT-I killing was evaluated by comparing the ratio of LLC-eGFP/OVA (target) over LLC-Kat (non-target) cells via flow cytometry. The percentage of specific killing normalized for the ratio in the cultures without OT-I cells was calculated with the following equation: 1−(%LLC-eGFP/OVA/%LLC-Kat) with CTLs/(%LLC-eGFP/OVA/%LLC-Kat) without CTLs. For spatiotemporal follow-up of target LLC-eGFP/OVA cell killing, eGFP intensity was monitored using Incucyte live cell imaging.
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