Five human T-ALL cell lines (Loucy, Jurkat, CCRF-CEM, KOPT-K1, and DND-41) were obtained from ATCC (Manassas, USA) and maintained in RPMI-1640 medium (Sigma, Saint-Louis, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 mg/mL streptomycin, and 100 U/mL penicillin at 37°C with 5% CO 2 . Human primary peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers from our hospital as previously described [25] and cultured in RPMI-1640 medium (Sigma) supplemented with 10% FBS. Human BMSCs (Shanghai Institute of Biological Sciences, Shanghai, China) were cultured in α-MEM (Sigma) containing 1% antibiotics and 10% FBS at 37°C with 5% CO 2 . BMSCs subjected to osteogenic differentiation were cultured in mineralization medium containing β-glycerophosphate (10 mmol/L), dexamethasone (10 -8 mol/L), and vitamin C (50 mg/L) for 14 days. The medium was changed every three days.
Rpmi 1640 medium
Manufactured by Merck Group
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RPMI-1640 is a widely used cell culture medium formulation, developed at Roswell Park Memorial Institute. It is a complete and balanced medium designed to support the growth and maintenance of a variety of cell types, including human and animal cell lines. The medium contains essential nutrients, vitamins, amino acids, and other components necessary for cell proliferation and survival in in vitro cell culture applications.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (616 mentions)
Fetal bovine serum (fbs), by Merck Group (537 mentions)
Streptomycin, by Merck Group (354 mentions)
Penicillin, by Merck Group (346 mentions)
L glutamine, by Merck Group (263 mentions)
Penicillin, by Thermo Fisher Scientific (241 mentions)
Streptomycin, by Thermo Fisher Scientific (238 mentions)
Penicillin streptomycin, by Merck Group (210 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (198 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (134 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (129 mentions)
L glutamine, by Thermo Fisher Scientific (127 mentions)
Fetal calf serum (fcs), by Merck Group (91 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (74 mentions)
Dmem medium, by Merck Group (57 mentions)
Hepes, by Merck Group (55 mentions)
Mcf 7, by American Type Culture Collection (52 mentions)
Phosphate buffered saline (pbs), by Merck Group (47 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (46 mentions)
Glutamax, by Thermo Fisher Scientific (46 mentions)
3 704 protocols using rpmi 1640 medium
1
Establishing Human T-ALL and BMSC Cultures
2
HIV-1 Subtype Infection in PBMCs
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PHA-stimulated PBMCs and lymphoblastoid cells were infected with HIV-1 (0.001 TCID50/cell) subtypes: A6 (GenBank: BankIt2701146 VSMO71 OQ979188), CRF02_AG (GenBank: MH062101.1), and B (GenBank: BankIt2701146 VSMO78 OQ979189) from the I.I. Mechnikov Institute of Vaccines and Sera HIV-1 isolates panel. Prior to infection, PBMCs at a concentration of 3 million/mL were aliquoted in a medium without serum for subsequent infection with each of the three HIV-1 subtypes (in triplicate). After 2 h of T-cell/PBMC + virus incubation at 37 °C in 5% CO2, the cells were washed twice with 1× phosphate-buffered saline (PBS). The cell pellets were resuspended in RPMI-1640 medium supplemented with 15% FBS (Sigma-Aldrich, Darmstadt, Germany, F9665). PBMCs and T-cells were cultured for 7 days in RPMI-1640 medium supplemented with 15% FBS (Sigma-Aldrich, Darmstadt, Germany, F9665), 100 U/mL of interleukin-2 (Sigma-Aldrich, Darmstadt, Germany, H7041), 2 mM glutamine, 100 U/mL of penicillin, and 100 U/mL of streptomycin. Out of the nine PBMCs isolated from female donors and infected with various subtypes of HIV-1, high viral production was achieved in PBMCs from five donors (no measurable p24 levels or low replicative activity were detected when PBMCs from the other four donors were infected with HIV-1).
3
Isolation and Cryopreservation of PBMCs
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PBMCs were isolated by density gradient centrifugation over Lymphoprep™ (Stem cell Technologies, Cat #07861) within 24 hours of collection. Peripheral blood was diluted 1:1 with RPMI-1640 Medium (Sigma, Cat#R8758) with 1% Penicillin/Streptomycin (Gibco™ Cat#15070063), layered at a ratio of 2:1 and centrifuged at 2000 x g for 25 minutes (brake off). Buffy coats were harvested and resuspended in RPMI media and centrifuged at 500 x g for 10 minutes and the number of cells determined using a haemocytometer (Fast-Read 102®, Kova International, Cat #88010). Cells were washed and centrifuged at 500 x g for 10 minutes before being cryopreserved in freezing media (RPMI-1640 Medium [Sigma, Cat#R8758], 10% DMSO [Sigma, Cat #D2650]).
4
Cultivating Cancer and Fibroblast Cell Lines
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Cell lines, namely LNCaP, PC-3, T47-D and NHDF were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in 75 cm2 culture flasks at 37 °C in a humidified air incubator with 5% CO2. LNCaP, PC-3 and T47-D cells, which were used in passages 20th to 27th, 25th to 29th and 10th to 15th, respectively, were cultured in RPMI 1640 medium (Sigma-Aldrich, Inc., St. Louis, MO, USA) with 10% fetal bovine serum (FBS; Sigma-Aldrich, Inc. St. Louis, MO, USA) and 1% of the antibiotic mixture of 10,000 IU/mL penicillin G and 100 mg/mL of streptomycin (Sp, Sigma-Aldrich, Inc. St. Louis, MO, USA). Finally, NHDF cells (Normal Human Dermal Fibroblasts) were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 1% of an antibiotic/antimycotic mixture (10,000 U/mL penicillin G, 100 mg/mL streptomycin and 25 µg/mL amphotericin B) (Ab; Sigma-Aldrich, Inc. St. Louis, MO, USA), and these cells were used in passages 10 to 12. For all cell types, the medium was renewed every 2–3 days until cells reach nearly the confluence state. Then, they were detached gently by trypsinization (trypsin-EDTA solution: 0.125 g/L of trypsin and 0.02 g/L of EDTA).
5
Stepwise Differentiation of hESCs into Hepatoblasts
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The differentiation protocol for the induction of DE cells and hepatoblasts was based on our previous report with some modifications [13–16–21]. Briefly, hESCs were dissociated by using dispase and suspended in MEF-conditioned ReproStem medium supplemented with 10 ng/ml FGF2, and then plated onto a growth factor reduced Matrigel (BD Biosciences)-coated dish. When hESCs reached approximately 80% confluence, the MEF-conditioned ReproStem medium was replaced with the differentiation RPMI-1640 medium (Sigma) containing 100 ng/ml Activin A (R&D systems) (the differentiation RPMI-1640 medium is consisted with RPMI-1640 medium (Sigma) supplemented with B27 supplement (Invitrogen) and 4 mM L-glutamine), and then cultured for 4 days. For induction of the hepatoblasts, the DE cells were cultured for 5 days in the differentiation RPMI-1640 medium supplemented with 20 ng/ml BMP4 (R&D Systems) and 20 ng/ml FGF4 (R&D Systems).
6
Melanoma Cell Lines Genetic Manipulation
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Melanoma cell lines SK-Mel-28 and 501Mel were grown in RPMI 1640 medium (Sigma) supplemented with 10% FCS. 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS and penicillin/streptomycin (7.5 μg/ml). Hermes-3A cells were grown in RPMI 1640 medium (Sigma) supplemented with 10% FCS, 200 nM TPA, 200 pM cholera toxin, 10 ng/ml human stem cell factor (Invitrogen), 10 nM endothelin-1 (Bachem, Bubendorf, Switzerland), and penicillin/streptomycin (7.5 μg/ml). All lentiviral shRNA vectors were obtained from Sigma (Mission sh-RNA series) in the PLK0 vector. The following constructs were used. shBRG1 (TRCN0000015549) and shMITF (TRCN0000019119). In each case between 5 × 105 and 1 × 106 cells were infected with the indicated shRNA lentivirus vectors and all experiments were performed at least in triplicate. siRNA knockdowns were performed with the corresponding ON-TARGET-plus SMARTpools purchased from Dharmacon Inc. (Chicago, Il., USA). Control siRNA directed against luciferase was obtained from Eurogentec (Seraing, Belgium). siRNAs were transfected using Lipofectamine RNAiMax (Invitrogen).
7
GEN2.2 pDC Cell Line Cultivation
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The human pDC cell line (GEN2.2) [102 (link)] used in our experiments was provided by Dr. Joel Plumas and Dr. Laurence Chaperot (Research and Development Laboratory, French Blood Bank Rhône-Alpes, Grenoble, France) and was deposited with the CNCM (French National Collection of Microorganism Cultures) under the number CNCMI-2938. GEN2.2 cells were grown on a layer of mitomycin C (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. M4287)-treated murine MS5 feeder cells (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, Cat. No. ACC 441) in RPMI 1640 medium (Sigma-Aldrich, Cat. No. R8758) supplemented with 10% heat-inactivated FBS (Life Technologies Corporation, Carlsbad, CA, USA, Cat. No. 10270-106), 100 U/mL penicillin, 100 μg/mL streptomycin (both from Biosera, Nuaille, France, Cat. No. XC-A4122/100) and 5% non-essential amino acids (Life Technologies Corporation, Cat. No. 11140050). For experiments, the GEN2.2 cells were removed from the feeder layer and seeded on 24-well plates at a concentration of 5 × 105 cells/500 μL in complete RPMI 1640 medium (Sigma-Aldrich). Cell lines were grown and incubated at 37 °C in a 5% CO2 humidified atmosphere.
8
Candida and Saccharomyces Isolates Characterization
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Candida and S. cerevisiae isolates and strains used in this study are listed in Table S1. The C. auris isolates 470121 and 470140 were a generous gift from Sarah Kidd (National Mycology Reference Center, Adelaide), and the other isolates were obtained from the CDC (Atlanta, USA). All isolates were maintained on YPD plates (1% yeast extract, 2% peptone, 2% glucose, and 2% agar). When precultures were required, single colonies of C. auris were picked from YPD plates, inoculated into liquid YPD medium, and grown at 30°C for 20 h. Unless stated otherwise, all experiments were carried out in serum-free RPMI 1640 medium (R6504, Sigma), buffered with 3.5% MOPS (morpholinepropanesulfonic acid), and adjusted to pH 7. For experiments investigating different C-sources, RPMI 1640 medium (R1383, Sigma) with 3.5% MOPS was used supplemented with glucose (0, 10, 20 mM) or mannose (10 mM). Growth experiments with petite-positive and petite-negative yeasts were carried out in YNB supplemented with 0.2% yeast synthetic dropout mix.
9
Isolation and Staining of Neutrophils
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Peripheral blood was drawn from healthy donors using Safety-Multifly-cannula (0.8 × 19 mm), safety-cannula (0.9 × 38 mm), and S-Monovettes containing lithium heparin (Li-Hep (16 IE/mL blood); all equipment above is from Sarstedt AG & Co, Nümbrecht, Germany) according to a protocol approved by the ethics committee (No.15-101-0043) at the University of Regensburg (Germany). PMNs were isolated using density gradient centrifugation based on the protocol provided by pluriSelect Life Science using LeukoSpin and LymphoSpin solutions (pluriSelect Life Science, Leipzig, Germany) [23 ]. The cells were then resuspended in RPMI 1640 medium (Sigma Aldrich, Steinheim, Germany) at a concentration of 18 × 106 cells/mL. PMNs were stained using 4′,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich Chemie Ltd., St. Louis, USA) to mark extracellular DNA and dihydrorhodamine-123 (DHR-123, Molecular Probes, Inc., Eugene, USA) to detect reactive oxygen species (ROS). Both markers were initially diluted with Dimethylformamide (DMF) and Dulbecco's phosphate-buffered saline (DPBS, both from Sigma Aldrich, Steinheim, Germany), followed by RPMI 1640 medium with 10% fetal calf serum (FCS, Sigma Aldrich, Steinheim, Germany) to reach their final concentrations of 50 ng/mL and 10 nM, respectively.
10
Transwell-Based Invasion Assay for Cancer Cells
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The invasion assay was performed using Transwell plates (pore size, 8 µm) with a Boyden chamber (Sigma-Aldrich; Merck KGaA). Transfected cells were washed twice with serum-containing RPMI-1640 medium (Sigma-Aldrich; Merck KGaA). A total of 1×105 T24 and SW780 cells were seeded onto the Transwell apparatus. Each insert was preloaded with Matrigel (50 µg; Sigma-Aldrich; Merck KGaA). Cells were suspended in 100 µl RPMI-1640 serum-free medium (Sigma-Aldrich; Merck KGaA) and placed in the top chambers. RPMI-1640 medium (100 µl; Sigma-Aldrich; Merck KGaA) containing 10% fetal calf serum (Sigma-Aldrich; Merck KGaA) was added to the bottom chambers. The chambers were incubated for 24 h at 37°C with 5% FBS in the lower chamber. Cells on the upper layer were removed by cotton buds and then washed with PBS. The cells that had invaded into the lower chambers were fixed with methanol for 15 min (Sigma-Aldrich; Merck KGaA) and stained with 5% crystal violet for 30 min at 20°C (Sigma-Aldrich; Merck KGaA). The results were visualized by light microscopy (DFC500; Leica Microsystems GmbH, Wetzlar, Germany) and the final values represent the mean from three fields on the membrane. The results were visualized at magnification, ×100. Experiments were performed in triplicate.
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