Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), phosphate buffer saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 1M), L-glutamine (100X), non-essential amino acids (100X), and antibiotics (Pen, Strep, Amphotericin B, 100X) were purchased from Thermo Fisher Scientific (GibcoTM, Waltham, MA, USA). All the cell culture dishes were purchased from Corning. Microscopic glass cover slips were purchased from Blue Star. Molecular grade paraformaldehyde, triton X-100, and 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) were purchased from Thermo Fisher Scientific. ZORBAX RRHD Eclipse Plus C18 column with 1.8 µ particle size, 2.1 mm inner diameter, and length 100 mm were purchased from Agilent Technologies, Santa Clara, CA, USA. Anti-actin and anti-HCV core antibodies were purchased from BIO-RAD (Hercules, CA, USA) and GeneTex (Irvine, CA, USA) respectively. Anti-dsRNA antibody was purchased from Millipore (Merk-Sigma, Burlington, MA, USA).
Non essential amino acids 100x
Manufactured by Thermo Fisher Scientific
Sourced in United States
Non-Essential Amino Acids 100X is a concentrated solution of non-essential amino acids. It is commonly used as a supplement in cell culture media to promote cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Anti dsrna antibody, by Merck Group (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Zorbax rrhd eclipse plus c18 column, by Agilent Technologies (1 mentions)
4 4 difluoro 1 3 5 7 8 pentamethyl 4 bora 3a 4a diaza s indacene bodipy, by Thermo Fisher Scientific (1 mentions)
Anti hsp90, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640, by Euroclone (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Hek293t, by Merck Group (1 mentions)
Non essential amino acids 1x, by Thermo Fisher Scientific (1 mentions)
Glutamax 1x, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin 1x, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Mccoy s 5a modified media, by Thermo Fisher Scientific (1 mentions)
5 protocols using non essential amino acids 100x
1
Immunofluorescence Microscopy Protocol
2
RNA Interference and Western Blot Analysis
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HPAF-II, Capan-1 and PANC-1 cells were grown in RPMI 1640 (Euroclone), MiaPaCa-2 cells were grown in DMEM (Sigma-Aldrich). All media were supplemented with 10% fetal bovine serum (FBS, Gibco), Non-Essential Amino Acids 100x (Thermo Fisher Scientific), gentamycin (Aurogene), penicillin and streptomycin (Aurogene). For RNA interference, cells were transfected with the indicated siRNAs (Table S1 ) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) and harvested after 48 h for protein and RNA analyses. For western blot analyses, cell pellets were resuspended in RIPA buffer (Tris-HCl 50mM, NP40 1%, NaCl 150mM, Na-Deoxycholate 0.5%, EDTA 2mM, SDS 0.1%) supplemented with 2mM Na-orthovanadate, 0.5mM sodium fluoride, 1mM dithiothreitol and Protease-Inhibitor Cocktail (Sigma-Aldrich). Extracts were incubated 10 min on ice, sonicated for 5 s and centrifuged for 10 min at 13,000 rpm, 4°C. Supernatants were diluted in SDS-PAGE sample buffer, boiled for 10 min and analyzed as described.54 (link) Primary antibodies: anti-QKI (Santa Cruz Biotechnology sc-517305), anti-RBFOX2 (Bethyl A300-864A), anti-RBM47 (Sigma-Aldrich SAB2104562) and anti-HSP90 (Santa Cruz Biotechnology sc-13119).
3
Culturing Mouse Embryonic Stem Cells and Fibroblasts
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Bruce 4 (C57/B6 Strain) mouse embryonic stem cells (ESCs) and HEK293T were purchased from ATCC. Mouse Embryonic Fibroblasts (MEFs) were isolated as previously described [8 (link)]. Briefly, E13.5 C57BL/6 mouse embryos from 13.5 day-pregnant female mice were placed in (1X) PBS, the head and embryonic internal organs were dissociated from the abdominal cavity, then trypsinized and passed through a 10-ml syringe to produce single-cell suspensions, which were further expanded. MEFs and HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) Sigma-Aldrich, Cat. No. D6429) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), Penicillin/Streptomycin (100 ug/ml; Thermo Fischer Scientific, Cat. No. 15140122), 2mM GlutaMAX (Gibco #,Cat. No. 35-050-061), and 0.1 mM Non-Essential Amino Acids 100X (Gibco, Cat. No. 11140050), at 37°C and 5% CO2. Bruce4 ESCs were cultured in complete ESC medium [Knock Out MediumDMEM (Gibco, Cat. No. 10829018), 20% ES-tested FBS (Pansera, Cat. No. P30-2602), Penicillin/Streptomycin 1X (Thermo Fischer Scientific, Cat. No. 15140122), GlutaMAX 1X (Gibco, Cat. No. 35-050-061), and Non-Essential Amino Acids 1X (Gibco, Cat. No. 11140050)], with 20 ng/mL mLIF (Santa Cruz Biotechnology, sc-4378).
4
Colorectal Cancer Cell Line Maintenance
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Two colorectal cancer cell lines (DLD-1 p53+/+ and HCT116 p53+/+), both a gift from Prof. Vogelstein (John-Hopkins-University, Baltimore, MD) were maintained in McCoy´s 5A (modified) Media (Gibco) supplemented with 10% foetal bovine serum (FBS) (Sigma Aldrich), 1% penicillin-streptomycin (Biochrom), 1% Sodium pyruvate and 1% Non-Essential Amino acids (100X) (Gibco). For lentiviral particle production, HEK293T cells (Thermo Fisher) were maintained in Hyclone- Dulbecco’s Minimum Essential Media (DMEM, Gibco), supplemented with 10% FBS and 200 μM L-glutamine. All cell lines were kept in an incubator at 37°C in 5% CO2.
5
Tumor Supernatant Cytokine Profiling
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The tumor supernatants were recovered as follows: tumors (cut into15-20 mg weight fragments) were incubated 24h at 37 C in culture medium (RPMI 1640 Glutamax (Gibco, #61870-044) supplemented with 10% FBS (HyClone #CH30160.03), 1mM Sodium Pyruvate (Gibco, #11360-088), 10 ml Non-essential amino acids 100X (Gibco, #11140-068) and 1% of Penicillin/Streptomycin (Penicillin 10.000U/ml; Streptomycin 10.000 mg/ml, Gibco, #15140-163). After incubation, the supernatants were diluted 1:2 (v/v) in culture media (description above), filtered (pore size of 0.22 mm) and frozen in aliquots at -80 C. Human Milliplex Map kits (Human cytokine/chemokine Magnetic Bead panels I, II, III (Millipore, #HCYTOMAG, #HCYP2MAG, #HCYP3MAG) were purchased from Millipore and used according to manufacturer's recommendations. A multiplex Luminex assay was used to measure the following cytokines simultaneously in the supernatants: IL-1b, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IL-17F, IL-17A, IL-17E, IL-21, IL-22, IL-28A, IL-31, IL-33, IFN-g, TNF-a, TNF-b, GM-CSF in accordance with manufacturer guidelines (Millipore).
The detection limit was 16pg/ml for IL-33 and IL-21, and 4pg/ml for the other measured cytokines.
The detection limit was 16pg/ml for IL-33 and IL-21, and 4pg/ml for the other measured cytokines.
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