U2OS cells were grown in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, St Louis, MO, USA) supplemented with 10% FBS (Sigma‐Aldrich), 2 mm l ‐glutamine (Sigma‐Aldrich), and 100 units·mL−1 penicillin and 100 μg·mL−1 streptomycin (Sigma‐Aldrich). U2OS cells bearing a copy of the DR‐GFP, SA‐GFP, or EJ5‐GFP reporter systems were grown in standard DMEM described above supplemented with 1 μg·mL−1 puromycin (Sigma‐Aldrich).
2 mml glutamine
Manufactured by Merck Group
Sourced in United States
2 mml-glutamine is a laboratory reagent used in cell culture applications. It serves as a source of the amino acid glutamine, which is an essential nutrient for many cell types. This product is provided in a concentrated liquid form to facilitate addition to cell culture media.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Penicillin streptomycin, by Euroclone (1 mentions)
Sodium pyruvate, by Euroclone (1 mentions)
Non essential amino acids (neaa), by Euroclone (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Lympholyte, by Cedarlane (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Human umbilical vein endothelial cells (huvec), by Thermo Fisher Scientific (1 mentions)
Medium 200prf, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Mml glutamine, by Merck Group (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Dmem f12, by Merck Group (1 mentions)
7 protocols using 2 mml glutamine
1
U2OS Cell Culture and Reporter Lines
2
Peripheral Blood Analysis of CHC, SLE, and HD
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Analyses were performed on PB samples from 15 CHC patients belonging to a previously described cohort, in which PB was collected at day 0 (before therapy) and at day 2 of therapy with pegIFN/ribavirin.16 PB was obtained from 21 SLE patients, whose clinical characteristics are described in Table 1 . Buffy coats, anonymously provided by the Immunohematology and Transfusion Center of Policlinico Umberto I, were used to obtain PB from HDs. PBMCs were isolated by density gradient centrifugation through Lympholyte (Cedarlane, Burlington, Canada) and collected in complete RPMI 1640 Dutch modified medium containing 10% FBS (HyClone GE Healthcare Life Sciences, Chicago, IL, USA); 2 mm l ‐glutamine (Sigma‐Aldrich, Saint‐Louis, MO, USA); penicillin/streptomycin, nonessential amino acids, sodium pyruvate (all from EuroClone, Pero, Italy); and 50 μm 2‐mercaptoethanol (Sigma‐Aldrich).
Human studies were performed in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and approved by the Institutional Ethical Committee (Prot. 120/16). Informed consent was obtained from all patients.
The ISG15 deficient patient was enrolled in a NIAID IRB‐approved protocol and provided written informed consent for study participation in accordance with the Helsinki Declaration. The study of human subjects was also approved by the IRB of Icahn School of Medicine (New York, USA; IF2349568).
Human studies were performed in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and approved by the Institutional Ethical Committee (Prot. 120/16). Informed consent was obtained from all patients.
The ISG15 deficient patient was enrolled in a NIAID IRB‐approved protocol and provided written informed consent for study participation in accordance with the Helsinki Declaration. The study of human subjects was also approved by the IRB of Icahn School of Medicine (New York, USA; IF2349568).
3
Culture of Malignant and Non-Malignant T Cell Lines
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The malignant T cell line MyLa2059 and PB2B and the non-malignant T cell lines MyLa1850 and MySi were generated from patients with MF [40 (link)-42 (link)]. Mac2a is a malignant anaplastic large-cell lymphoma (ALCL) cell line established from a skin tumor in the progressive phase of the disease [43 (link)] The Jurkat T cell line, J-Tag [44 (link)], and the B-cell line, Ramos 2G6 [45 (link)], have been described elsewhere. MyLa2059, PB2B, Mac-2a and Ramos were grown in conditional media (CM) (RPMI 1640, 2 mm l-glutamine and 100 g/ml penicillin/streptomycin; all from Sigma) supplemented with 10% fetal bovine serum (Life Technologies, Denmark). The MyLa1850 and MySi cell lines were grown in CM supplemented with 10% pooled human serum (HS) (Blood Bank, State University Hospital, Copenhagen, Denmark) and 103 U/ml interleukin (IL)-2 (Proleukin™). Primary human umbilical vein endothelial cells (HUVEC), pooled from multiple donors, were purchased from Life Technologies, Denmark. All experiments were performed with cells at passage 2 to 4. HUVEC cells were cultured in Medium 200PRF (Life Technologies, Denmark) supplemented with low serum growth supplement (LSGS) (Life Technologies, Denmark).
4
Isolation and Culture of Immune Cells from RA Patients
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Synovial fibroblasts were isolated from RA patients (n = 3) undergoing knee replacement surgery at Karolinska
University Hospital and cultured in complete media consisting of high-Glc DMEM,
10% heat-inactivated FCS, 100 µg/ml penicillin–streptomycin and 2 mM
L -glutamine from Sigma-Aldrich (Saint Louis, MO, US) in standard tissue
culture (TC) flasks (Sarstedt, Nümbrecht, Germany) in a TC incubator at 37°C
with 5% CO2. Chondrocytes were isolated from RA patients (n = 3) undergoing knee replacement surgery at
Karolinska University Hospital. Chondrocytes were cultured in complete media
with DMEM-F12 (Sigma-Aldrich) in standard TC flasks (Sarstedt) in a TC incubator
at 37°C with 5% CO2. PBMCs were purified from blood samples from
healthy adult donors (n = 3) using standard
Ficoll-Paque (Ficoll-Paque Plus, GE Healthcare, Uppsala, Sweden) separation
(www.miltenyibiotec.com ). Washed cells were resuspended in
Roswell Park Memorial Institute-1640 media supplemented with 10%
heat-inactivated FCS, 100 µg/ml penicillin–streptomycin and 2 mM
L -glutamine (Sigma-Aldrich). All procedures were approved by the
Institutional Ethical Committee (Solna, Stockholm, Sweden, ethical number
2009/1262-31/3) and are in compliance with all ethical standards and patients’
written consent, in accordance with the Declaration of Helsinki.
University Hospital and cultured in complete media consisting of high-Glc DMEM,
10% heat-inactivated FCS, 100 µg/ml penicillin–streptomycin and 2 mM
culture (TC) flasks (Sarstedt, Nümbrecht, Germany) in a TC incubator at 37°C
with 5% CO2. Chondrocytes were isolated from RA patients (n = 3) undergoing knee replacement surgery at
Karolinska University Hospital. Chondrocytes were cultured in complete media
with DMEM-F12 (Sigma-Aldrich) in standard TC flasks (Sarstedt) in a TC incubator
at 37°C with 5% CO2. PBMCs were purified from blood samples from
healthy adult donors (n = 3) using standard
Ficoll-Paque (Ficoll-Paque Plus, GE Healthcare, Uppsala, Sweden) separation
(
Roswell Park Memorial Institute-1640 media supplemented with 10%
heat-inactivated FCS, 100 µg/ml penicillin–streptomycin and 2 mM
Institutional Ethical Committee (Solna, Stockholm, Sweden, ethical number
2009/1262-31/3) and are in compliance with all ethical standards and patients’
written consent, in accordance with the Declaration of Helsinki.
5
Cell Line Maintenance Protocol
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The mouse fibroblast L929, Madin-Darby Canine Kidney (MDCK), and Human embryonic
kidney 293T (HEK 293T) cell lines were procured from the National Centre for
Cell Science, Pune, India, and maintained in Dulbecco’s modified Eagle’s tissue
culture medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10%
fetal calf serum, 100 units/ml penicillin, and 100 units/ml streptomycin in
tissue culture flasks (Corning, USA) at 37°C in a CO2 incubator. Cell
lines grown to monolayer cultures were maintained in Dulbecco’s modified Eagle’s
medium (Gibco, Thermo Fisher Scientific, USA) supplemented with heat-inactivated
10% fetal bovine serum (Gibco, Thermo Fisher Scientific, USA) and 2 mM
L-glutamine (Sigma-Aldrich, India), 100 units/ml penicillin and 100 μg/ml
streptomycin and maintained at 37°C in an atmosphere of 5% CO2incubator at 95% air humidified. The cultivated cells were regularly controlled
for cell growth and the absence of mycoplasmas.
kidney 293T (HEK 293T) cell lines were procured from the National Centre for
Cell Science, Pune, India, and maintained in Dulbecco’s modified Eagle’s tissue
culture medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10%
fetal calf serum, 100 units/ml penicillin, and 100 units/ml streptomycin in
tissue culture flasks (Corning, USA) at 37°C in a CO2 incubator. Cell
lines grown to monolayer cultures were maintained in Dulbecco’s modified Eagle’s
medium (Gibco, Thermo Fisher Scientific, USA) supplemented with heat-inactivated
10% fetal bovine serum (Gibco, Thermo Fisher Scientific, USA) and 2 mM
L-glutamine (Sigma-Aldrich, India), 100 units/ml penicillin and 100 μg/ml
streptomycin and maintained at 37°C in an atmosphere of 5% CO2incubator at 95% air humidified. The cultivated cells were regularly controlled
for cell growth and the absence of mycoplasmas.
6
Culturing N9 Microglial and MES 23.5 Dopaminergic Cells
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The murine N9 microglial cell line was provided by Dr Paola Ricciardi-Castagnoli (Singapore Immunology Network, Agency for Science, Technology and Research, Singapore). The N9 microglial cells were cultured in Roswell Park Memorial Institute medium (RPMI 1640; Invitrogen, 21,875-091) supplemented with 10% FBS, 2mML-Glutamine (Sigma, G6392),100 U/ml penicillin, and 100 mg/ml streptomycin, and the cultures were maintained at 37°C, 95% air, and 5% CO2 in a humidified incubator [67 (link)]. The cells were then seeded onto 35-mm culture dishes (0.5 x106 cells/ well) for analysis.
Dopaminergic MES 23.5 cells, a gift from Dr Wei-dong Le (Baylor College of Medicine, TX, USA), were cultured in DMEM/F12 containing Sato components growth medium supplemented with 2% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified CO2 incubator (5% CO2, 95% air) [68 (link)]. For experiments, MES 23.5 cells were plated at a density of 0.5 × 105/cm2 onto 35-mm plastic dishes, glass coverslips, previously coated with poly-L-ornithine (P-4638, Sigma; 10 mg/ml). Cells were stimulated to enhance differentiation by adding dibutyryl-cAMP (D0627, Sigma; 1 mM) to the supplemented growth medium, and they were grown to 80% confluence before the start of any treatment.
Dopaminergic MES 23.5 cells, a gift from Dr Wei-dong Le (Baylor College of Medicine, TX, USA), were cultured in DMEM/F12 containing Sato components growth medium supplemented with 2% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified CO2 incubator (5% CO2, 95% air) [68 (link)]. For experiments, MES 23.5 cells were plated at a density of 0.5 × 105/cm2 onto 35-mm plastic dishes, glass coverslips, previously coated with poly-L-ornithine (P-4638, Sigma; 10 mg/ml). Cells were stimulated to enhance differentiation by adding dibutyryl-cAMP (D0627, Sigma; 1 mM) to the supplemented growth medium, and they were grown to 80% confluence before the start of any treatment.
7
Comprehensive Cell Culture Reagents
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DMEM cell culture medium was purchased from Gibco. Fetal bovine serum, 2 mM l-glutamine and PEST, trypsin-EDTA, bovine serum albumin, 37% formaldehyde, formamide, glycerol, sodium citrate Buffer were purchased from Sigma. Secure-Seal hybridization chambers were purchased from Invitrogen. Superfrost Plus slides and Uracil-DNA glycosylase were purchased from Thermo Fischer Scientific. All oligonucleotides were purchased from Integrated DNA Technologies. Petri dishes were purchased from Corning. TranscriptMe, RiboLock RNase Inhibitor, RNase H, T4 ligase, deoxyribonucleotide triphosphate, adenosine triphosphate were ordered from DNA Gdansk. Ampligase was purchased from Epicentre, Illumina. phi29 DNA polymerase was purchased from Monserrate. DAPI was purchased from Biotum.
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