293T cells were obtained through NIH AIDS Research and Reference Reagent Program. 293T cells were cultured in DMEM medium supplemented with 10% FBS and 100 I.U./mL penicillin/100 μg/mL streptomycin solution (all from Invitrogen). Jurkat, CEM (all purchased from ATCC) and PBMCs were cultured in RPMI medium supplemented with 10% FBS and 100 I.U./mL penicillin/100 μg/mL streptomycin solution (all from Invitrogen). PBMCs were isolated from whole healthy donor blood on a 1.077 g/cm3 Ficoll-Paque density gradient (PanEco, Russia), washed twice using PBS supplemented with 2% FBS and cultured in RPMI culture medium. Cells were activated with 1 µg/mL PHA (Sigma) for 2 days and grown in the presence of 100 U/mL of recombinant human interleukin-2 (Ronkoleikin, Biotech, Russia). The proportion of CD4+ cells in isolated PBMCs was equal to 63 ± 10% according to the flow cytometry analysis, performed on FACS Canto II (BD, USA) using APC-Cy7-conjugated anti-CD3 (clone SK7, BD PHArmingen, USA) and APC-conjugated anti-CD4 (clone RPA-T4, BD PHArmingen, USA) antibodies. All experiments with the human blood samples were approved by the Human Ethics Committee of the Institute of Immunology (Moscow), and blood donors gave informed consent for the use of their samples in the described experiments. All methods were performed in accordance with relevant the guidelines and regulations.
Streptomycin solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Canada, Japan
Streptomycin solution is a sterile, concentrated aqueous solution of the antibiotic streptomycin sulfate. It is a broad-spectrum antibiotic commonly used in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (36 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
Hepes, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Spf embryonated chicken eggs, by Charles River Laboratories (3 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Curcumin, by Merck Group (2 mentions)
Atcc ccl 34, by American Type Culture Collection (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
40 protocols using streptomycin solution
1
Isolation and Activation of Primary Human T Cells
2
TNBC Cell Line Culturing Protocol
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Human TNBC cell lines MDA-MB-231 and SUM-159 and non-tumor breast epithelial cell MCF-10A were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The TNBC cells (MDA-MB-231 and SUM-159) were cultured at 37℃ with 5% carbon dioxide in medium (MDA-MB-231: DMEM medium and SUM-159: RPMI 1640 medium) supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin solution (Gibco Life Technologies, Lofer, Austria). MCF-10A was cultured in Dulbecco’s modified Eagle medium containing 5% horse serum, 100 U/mL penicillin, and 100 μg/mL streptomycin solution (Gibco Life Technologies, Gaithersburg, MD), 20 ng/mL recombinant human epidermal growth factor, 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin and 10 μg/mL insulin (Sigma-Aldrich, Shanghai, China).
3
Primary Cell Culture Establishment
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The primary culture was established using previously established protocols.[5 6 (link)] Briefly, tissue was minced into <1 mm length using a sterile surgical blade and scalpel. The tissue pieces were incubated in 0.1% collagenase type IV enzyme (Gibco-Invitrogen) for 1 h and then the enzyme digested tissue sample was centrifuged at 1000 rpm for 5 min and the supernatant was discarded. Later, 4–5 tissue explants were placed in culture dish overnight, with a minimum amount of Dulbecco’s modified Eagle’s medium (DMEM)-High glucose (Gibco-Invitrogen) with 20% fetal bovine serum (FBS, (Gibco-Invitrogen), 100 U/mL penicillin and 100 µg/mL streptomycin solution (Gibco-Invitrogen). The culture was continued till the cells reached 60%–70% confluence.
4
Mouse Ovarian Surface Epithelial Cell Culture
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The animal protocol for this study was approved by the Institutional Animal Care and Use Committee (IACUC) of Fuzhou General Hospital (Fujian, China) and followed the regulatory animal care guidelines of the United State National Institute of Health (Bethesda, MD, USA). In this study, we obtained six-month female BALB/c mice from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). We isolated mouse ovarian surface epithelial cells (MOSECs) and cultured them in the “MOSE medium” containing α-Minimum Essential Medium from Thermo-Fisher Scientific Company (Waltham, MA, USA) supplemented with 4% heat-inactivated 3:1 donor bovine serum: fetal bovine serum (Gibco, Gaithersburg, MD, USA), 5 U/ml of penicillin and 5 µg/ml of streptomycin solution (Invitrogen, Carlsbad, CA), 0.1 µg/ml of gentamicin (Invitrogen) at 37 °C with 5% CO2 according to the previous described protocols (Gamwell, Collins & Vanderhyden, 2012 (link); McCloskey et al., 2014 (link)). The MOSECs with spontaneously malignant transformation phenotypes were maintained in the MOSE medium according to a previous study (Flesken-Nikitin et al., 2013a (link)), which have two phases, i.e., the early stage (MOSE-I; normal epithelial cells) and the later stage (MOSE-II; various phenotypes of ovarian cancer cells).
5
Isolation and Stimulation of PBMCs
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PBMCs were separated from 20 mL of heparinized whole blood from AR children. Specifically, cells were isolated by Lymphoprep (Fresenius Kabi Norge AS, Oslo, Norway) density-gradient centrifugation from heparinized leucocyte-enriched buffy coats. Then, PBMCs were cultured at 2∗106/mL in 24-well plates in culture medium: RPMI 1640 supplemented with 5% human AB serum, 5 mmol/L glutamine, and penicillin, and streptomycin solution (all from Invitrogen, except for serum from Sigma-Aldrich). Stimulation was performed through addition of rhIL-37b (1–100 ng/mL) with or without other cytokines (Poly (I:C) (1 ng/mL), rhIL-4 (1–100 ng/mL), rhIL-5 (1–100 ng/mL), rhIL-13 (1–100 ng/mL), rhIL-12 (1–100 ng/mL), rhIFN-γ (1–100 ng/mL), rhIL-10 (1–100 ng/mL), SB203580 (10 μmol/L), and LY294002 (10 μmol/L). All the above stimulators were from R&D systems.
6
Bacterial Cell Culture and Staining Protocols
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Tryptic soy agar (TSA, w/5% sheep blood) plates, tryptic soy broth (TSB), phosphate buffered saline (PBS), fetal bovine serum (FBS), 0.25% trypsin/2.21 mM ethylenediaminetetraacetic acid (EDTA) solution, 45% glucose solution, 7.5% sodium bicarbonate, sodium pyruvate, and HEPES buffer were all obtained from Fisher Scientific (Pittsburgh, PA). Dulbecco’s Modified Eagle Media: Nutrient Mixture F-12 (DMEM/F12) and RPMI-1640 media were purchased from LONZA (Walkersville, MD). DiI fluorescent dye, Syto-9, propidium iodide (PI), and 100 U/mL penicillin/100 mg/mL streptomycin solution were from Invitrogen (Carlsbad, CA). Gentamicin, Triton X-100, cytochalasin D, dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), lysostaphin, fluorescein isothiocyanate (FITC), paraformaldehyde, and glutaraldehyde were obtained from Sigma (St. Louis, MO). Primary antibody Ab20920 and goat polyclonal to rabbit IgG secondary antibody-Cy5 ab6564 were obtained from Abcam (Cambridge, MA). Dichlorofluorescin diacetate (H2DCF-DA) and dihydroethidum (DHE) were from Life Technologies (Grand Island, NY).
7
Cell Culture Conditions for Cancer Cell Lines
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HSC-3 and Ca9-22 cells were obtained from the RIKEN CELL BANK (Tsukuba, Japan). UMSCC-81 cells were kindly provided by the University of Michigan. These cells were cultured in Dulbecco’s Modified Eagle’s Medium ( Nissui Pharmaceutical Company, Tokyo, Japan) supplemented with 10% fetal bovine serum (Gibco, Grand Island, New York), 100 U/mL of penicillin, and a 100 μg/mL streptomycin solution (Invitrogen Corp, Carlsbad, California) and incubated at 37°C in a 5% CO2 atmosphere.
8
Neuron Culture from Embryonic Mouse Cortex
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Neuron-enriched mouse cerebral cortical cultures were prepared from the brains of embryonic day-16 wild-type 129 and HINT1 knockout mice. Cerebral cortices were dissociated and seeded (1.25 × 105 cells/cm2) onto multiwell dishes coated with poly-D-lysine. After 3 h, the culture medium was changed to Neurobasal medium supplemented with B-27, GlutaMAX and antibiotics (100 IU/ml Penicillin and 100 µg/mL Streptomycin solution) (Invitrogen, Paisley, UK). From days 5–7 in vitro, cytosine arabinoside (5 μM) was added to the cultures to eliminate the majority of proliferating non-neuronal cells. Cultures were maintained at 37 °C in a humidified 5% CO2 incubator. In some cases, cells were evaluated after transfection for 72 h, with the concentrated lentiviral vector coding the HINT1 protein cDNA.
9
Murine Bone Marrow Dendritic Cell Culture
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Femurs and tibias were collected from front and back leg of donors. They were flushed with FACS buffer, erythrocytes were lysed with ACK for 3 minutes, and BM cells were then cultured with 300 ng/mL Flt3L (homemade) at 2 3 10 6 cells/mL in 10 mL of BM DC media (RPMI-1640 [Invitrogen] containing 10% heat-inactivated FCS, 100 U/mL penicillin, 100 mg/mL streptomycin solution [Invitrogen], and 200 mM L-glutamine [Invitrogen]) in a 10-cm petri dish. On day 3 and 5, 5 mL of fresh BM DC media with 300 ng/mL Flt3L was added.
10
Protein Expression Analysis in Cell Culture
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The cell culture medium DMEM, FBS, penicillin, streptomycin solution, and 25 mM HEPES reagents were purchased from Invitrogen (Carlsbad, CA, USA). The Lipofectamine 3000 reagent (Cat. #L3000015) was purchased from Thermo Fisher Scienti c (Waltham, MA, USA). Primary antibodies against PSMD4 (ab137109), Myc (ab32072), Tubulin (ab210797), Actin (ab8226), LaminB (ab133741), E-cadherin (ab1416), vimentin (ab92547), cyclin A (ab181591), cyclinB1 (ab32053), cyclin E (ab33911), CK-8 (ab53028), CK-18 (ab133263), and p53 (ab32389) were purchased from Abcam (Cambridge, UK). The GFP (GTX113617) antibody was purchased from GeneTex (Texas, USA). Antibodies PI3K (#4249T), p-PI3K (#17366S), PDK1 (#5662S), p-PDK1 (#3438T), Akt (#4691S), p-Akt (S473) (#4060S), p-Akt (S474) (#8599S), p-Akt (T308) (#13038T), GSK-3β (#4818S), p-GSK-3β (S9) (#5558T), ERK (#9102S), p-ERK (#4370T), mTOR (#2983T), p-mTOR (S2448) (#5536T), p-mTOR (S2481) (#2974S), 4E-BP1 (#9644T), p-4E-BP1 (T37/46) (#2855T), Cdc25C (#4688T ), p-Cdc25C (S216) (#4901T), Cdc2 (#9116T), and p-Cdc2 (T15) (#4539T) and COX2 (#12282T) were purchased from Cell Signaling Technology (Beverley, USA).
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