The human HepaRG cells were grown in William’s E medium (Sigma, St Louis, MO, USA) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, USA), 2 mM L-glutamine (Eurobio, Les Ulis, France), 5 μg/mL insulin (Sigma), 50 μM hydrocortisone hemisuccinate (Serb, Paris, France), 50 U/mL penicillin and 50 μg/mL streptomycin (Eurobio). HepaRG cells were used after 15 days post-plating. Human skin fibroblasts were obtained from biopsies of non-lesional skin as previously described8 (link). Fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (Eurobio) supplemented with 10% fetal bovine serum (Life Technologies), 2 mM L-glutamine (Eurobio), 100 U/mL penicillin and 100 μg/mL streptomycin (Eurobio).
L glutamine
Manufactured by Eurobio Scientific
Sourced in France, United States, Italy
L-glutamine is an amino acid that plays a crucial role in various metabolic processes within the body. It serves as an important source of nitrogen and is involved in the synthesis of proteins, nucleic acids, and other biomolecules. L-glutamine is naturally present in many foods and can also be obtained through supplementation.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Eurobio Scientific (18 mentions)
Streptomycin, by Eurobio Scientific (18 mentions)
Penicillin streptomycin, by Eurobio Scientific (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Dulbecco modified eagle medium (dmem), by Eurobio Scientific (7 mentions)
Sodium pyruvate, by Eurobio Scientific (7 mentions)
Amphotericin b, by Eurobio Scientific (7 mentions)
Rpmi 1640, by Eurobio Scientific (6 mentions)
Fetal bovine serum (fbs), by Eurobio Scientific (6 mentions)
Fetal calf serum (fcs), by Eurobio Scientific (4 mentions)
Rpmi 1640 medium, by Eurobio Scientific (3 mentions)
Hepes, by Eurobio Scientific (3 mentions)
Human ab serum, by Thermo Fisher Scientific (3 mentions)
Hepes buffer, by Eurobio Scientific (3 mentions)
Williams e medium, by Merck Group (2 mentions)
Insulin, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium, by Eurobio Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Eurobio Scientific (2 mentions)
Fetal bovine serum (fbs), by PAN Biotech (2 mentions)
42 protocols using l glutamine
1
Culturing HepaRG Cells and Human Skin Fibroblasts
2
HT29 and HT29-MTX Cell Culture Protocol
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The human adenocarcinoma cell line HT29 and the mucus-secreting HT29 cells selected by adaptation to methotrexate (HT29-MTX) were used. HT29-MTX cells were obtained from Dr Thécla Lesuffleur (INSERM UMR S 938, Paris France) (Lesuffleur et al., 1993 (link)). Cells were routinely grown in 25 cm2 plastic tissue culture flasks (Nunc, Life Technologies) in 5 ml of culture medium (Dulbecco's modified Eagle's medium; DMEM; Eurobio, Courtaboeuf, France) supplemented with 10% (v/v) fetal calf serum (Eurobio, Courtaboeuf, France), 2 mM L-glutamine (Eurobio, Courtaboeuf, France) and antibiotics—penicillin 100 IU ml−1 and streptomycin 100 μg ml−1 (Sigma, France). Antibiotics were routinely added to the culture medium except for virulence assays. Cells were maintained in a humidified incubator (at least 90% RH) at 37°C under 5% (v/v) CO2 (SANYO Electric Co., Ltd., Osaka, Japan). The medium was changed every other day.
3
UV Irradiation of A549 Cells
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A549 cells were cultured in DMEM (Eurobio) containing 10% FCS (Pan Biotech) and L‐Glutamine (Eurobio) at 37°C in 5% CO2. siRNA reverse transfections were performed in 10 cm with Lipofectamine RNAiMAX (Thermo Scientific) at a final concentration of 20 nM siRNA (Eurogentec or Dharmacon; see Appendix Table S1 ) as per the manufacturer's instructions in OptiMEM reduced serum media (Thermo Scientific). After 48 h transfection, cells were washed with PBS and irradiated with 40 J/m2 UV (254 nm; Stratalinker), placed in fresh media, and harvested on ice after 16 h of recovery at 37°C.
4
Isolation and Propagation of Metastatic Tumor Cells
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Metastatic cells were obtained as reported by Pulaski,27 with minor changes. BALB/c mice were inoculated with 1 × 104cells into the fourth mammary fat pad, allowing primary tumor development.
Twenty-five days later, the mice were humanely euthanized by CO2inhalation. First, the primary tumor, lungs, and liver were resected.
Subsequently, the organs and tumor were transferred to RPMI-1640 media (Eurobio)
supplemented with 2% penicillin/streptomycin (Eurobio). Next, the tissues were
sliced into small pieces and digested with 2.5 mL of type-IV collagenase (Gibco,
Life Technologies, New York, NY) in RPMI-1640 media supplemented with 10 mM
HEPES, 2.5% fetal bovine serum (FBS), 2% penicillin/streptomycin, 1 mM sodium
pyruvate, and 2 mM L-glutamine (Eurobio); lung and liver digestion was carried
out at 4°C for 90 minutes. Subsequently, the tissues were filtered by a 70 µm
cell strainer and centrifuged for 5 minutes at 300g, and after
the lysis of red blood cells, the cells were cultured in the presence of
6-thioguanine (Sigma-Aldrich, St. Louis, MO) to select metastatic tumor cells.
Once colonies were observed, attached cells were recovered with 0.25%
trypsin/0.02% EDTA (Eurobio), and then the cells were washed with supplemented
RPMI media and transferred to T-25 culture flasks. Metastatic cells were
obtained from at least 3 different mice during 3 serial in vivo passages.
Twenty-five days later, the mice were humanely euthanized by CO2inhalation. First, the primary tumor, lungs, and liver were resected.
Subsequently, the organs and tumor were transferred to RPMI-1640 media (Eurobio)
supplemented with 2% penicillin/streptomycin (Eurobio). Next, the tissues were
sliced into small pieces and digested with 2.5 mL of type-IV collagenase (Gibco,
Life Technologies, New York, NY) in RPMI-1640 media supplemented with 10 mM
HEPES, 2.5% fetal bovine serum (FBS), 2% penicillin/streptomycin, 1 mM sodium
pyruvate, and 2 mM L-glutamine (Eurobio); lung and liver digestion was carried
out at 4°C for 90 minutes. Subsequently, the tissues were filtered by a 70 µm
cell strainer and centrifuged for 5 minutes at 300g, and after
the lysis of red blood cells, the cells were cultured in the presence of
6-thioguanine (Sigma-Aldrich, St. Louis, MO) to select metastatic tumor cells.
Once colonies were observed, attached cells were recovered with 0.25%
trypsin/0.02% EDTA (Eurobio), and then the cells were washed with supplemented
RPMI media and transferred to T-25 culture flasks. Metastatic cells were
obtained from at least 3 different mice during 3 serial in vivo passages.
5
Expansion of Vγ9Vδ2 T cells in vitro
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PBMC were plated for 7 days at 106 cells per ml in 96-wells round-bottomed plates at 37 °C in a humidified atmosphere of 5% CO2 in air in the presence of 10 IU ml−1 IL-2 (Eurocetus, Milano, Italy) and 1 μM ZA (kindly provided by Novartis Pharma, Origgio, Italy). The standard culture medium was RPMI 1640 (Eurobio, Les Ulis, France), containing 10% FCS (Mascia Brunelli, Milano, Italy), 2 mM L -glutamine (Eurobio, Les Ulis, France), 100 U ml−1 penicillin (Eurobio, Les Ulis, France) and 100 mg ml−1 streptomycin (Eurobio, Les Ulis, France)3 (link). Total counts of viable Vγ9Vδ2 T cells on day 7 were evaluated by multiplying total counts of viable cells per well, identified with the trypan blue staining assay, by the percentage of Vγ9Vδ2 T cells identified by flow cytometry46 (link).
6
Synovial Cells from PVNS and RA Patients
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Synoviocytes were obtained from patients undergoing joint surgery (Hospitals Edouard Herriot and Croix Rousse, Lyon, France), with diagnosed PVNS (mean age: 37 years, ratio F/M: 2/1) or with RA (mean age: 62 years, ratio F/M: 5/1), who fulfilled the American College of Rheumatology criteria for RA51 (link). The synovial tissue was cut into small pieces and then adhered in 6 well-plates in Dulbecco’s modified Eagle’s medium (DMEM; Eurobio, Courtaboeuf, France) supplemented with 10% fetal bovine serum (FBS; Life Technologies, by Thermo Fisher Scientific, Carlsbad, USA), 2 mM l -glutamine (Eurobio) and 100 U/ml penicillin/streptomycin (Eurobio) (Complete DMEM). Cells were maintained at 37 °C in a humidified 5% carbon dioxide incubator and used between passages 4 to 9. Each patient signed an informed consent form. The protocol was approved by the Ethics Committee of the Hospitals of Lyon for the protection of persons participating in biomedical research under the number AC-2016-272.
7
Isolation and Culture of B-Cells for CLL Studies
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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. CD19+ CD5+ B-cells were isolated from PBMCs using magnetic-bead-activated cell sorting, using a B-CLL cell isolation kit (Miltenyi Biotec). To evaluate the effect of AA on normal lymphocytes, naïve B-cells were isolated from donor lymphocyte infusions using a Naïve B-cell Isolation kit (Miltenyi Biotech). The purity assessed by CD19 expression on flow cytometry was around 98%. OSU-CLL cells were a gift from E. Hertlein and colleagues [28 (link)]. JVM3 cells were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Freshly isolated B-cells and CLL cell lines were cultured in RPMI 1640 medium (PAN Biotech, #P04–16500) with 10% fetal bovine serum (FBS) (PAN Biotech, #P30–3306) and L-glutamine, penicillin and streptomycin (1%) (Eurobio Scientific). When indicated, CLL B-cells were cultured in Iscove’s modified Dulbecco’s Medium (IMDM) (Merck, #FG0465) and alpha-MEM medium (Sigma Aldrich, #M4526) (n = 7). Cells were cultured at a density of 4 × 105/ml in 48-well plates and were treated with either vehicle or AA or drugs for 24 h. To simulate hypoxia condition, cells were cultured in presence of 100 μM Cobalt(II) chloride hexahydrate (CoCl2.6H2O, Sigma Aldrich) for 24 h. Cells were then washed and incubated with AA for 24 h.
8
Isolation and Characterization of Mesenchymal Stem Cells from Human Follicular Fluid
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Human follicular fluids were collected as residual samples during the assisted reproductive techniques of oocyte collection from female ovaries by transvaginal ultrasound-guided aspiration using a single aspiration needle (Cook Medical, Bloomington, IN, USA) and phenotypically analysed to assess their mesenchymal properties (Figure S1 ) [15 (link)]. Mesenchymal stem cells present in the hFF were isolated by centrifuging the follicular fluid by density gradient separation (Lymphoprep, NycomedPharma, Oslo, Norway) for 30 min at 1800 rpm (to eliminate red blood cells and debris), as reported in [15 (link)]. hFF-MSCs were cultured at 37 °C in a humidified incubator with 5% CO2 in DMEM medium (Sigma-Aldrich) supplemented with 10% foetal bovine serum (FBS, Eurobio, Les Ulis, France), 2 mM of L-glutamine (Eurobio), 100 mg/mL of penicillin (Eurobio) and 100 μg/mL of streptomycin (Eurobio). Cells were used for all these experiments at the first passage of cell culture.
9
Culture and Maintenance of Leukemic Cell Lines
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The human leukemic cell line K562 (from a patient with chronic myeloid leukemia (CML)) was obtained from American type culture collection (Manassas, VA, USA). The human leukemic cells lines U937 (from a patient with monocytic differentiation-inducible histiocytic lymphoma) and Jurkat (from the peripheral blood of a 14-year-old boy with T-cell acute lymphoid leukemia) were provided by Dr. Hélène Moins‐Teisserenc (Laboratoire d'Hématologie Biologique, AP‐HP, Hôpital Saint Louis, 75010 Paris, France). Cells were grown in RPMI-1640 (Eurobio, Toulouse, France) supplemented with heat-inactivated fetal bovine serum (10%; Eurobio), 2 mM L-glutamine, 100 U/mL penicillin, 100 ug/mL streptomycin, 0.01 M Buffer Hepes, and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO2. Cell lines were proven mycoplasma-free using a MycoProbe Mycoplasma Detection Kit (R&D Systems) and maintained with ciprofloxacin (0.5 μg/mL).
10
Isolation and Culture of Synoviocytes from RA and OA Patients
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Synoviocytes were grown from fresh synovial tissue samples aseptically isolated from RA and osteoarthritis (OA) patients’ joints. The RA patients fulfilled the American College of Rheumatology criteria for RA [21 (link)]. All patients signed an informed consent form and the study was approved by the ethics committee of the hospitals of Lyon. The synovial tissue, minced in small pieces, was allowed to adhere to plastic plates and maintained in DMEM medium (Eurobio, Courtaboeuf, FR) supplemented with 10% FBS (Life Technologies by Thermo Fischer scientific, Grand Island, NY, USA), 2% Penicillin-Streptomycin, 1% L-glutamine and 1% Amphotericin B (all Eurobio) until cells reach 90% confluence. Synoviocytes were used between the fourth and ninth passages to ensure the cell specificity. All data are the results of at least three separate experiments (n = 3) and patients.
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