B16-F10, 4T1, Mel-Rel, TS/A, MCF-7, MDA-MB-468, LAU 145, and Me290 cell lines were cultured in RPMI-1640 (Eurobio, Toulouse, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO2. Cells were grown until 75% confluency and passaged using trypsin/1× EDTA (Eurobio), washed with PBS, and resuspended in supplemented RPMI-1640.
Rpmi 1640
Manufactured by Eurobio Scientific
Sourced in France
RPMI 1640 is a widely used cell culture medium formulated to support the growth of a variety of cells, including human and animal cells. It provides a balanced combination of essential nutrients, vitamins, and salts to maintain cell viability and proliferation in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
L glutamine, by Thermo Fisher Scientific (8 mentions)
Heat inactivated fbs, by Thermo Fisher Scientific (7 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (7 mentions)
Heat inactivated, by Thermo Fisher Scientific (7 mentions)
Tnf α, by R&D Systems (7 mentions)
Anti cd34 coated magnetic beads, by Miltenyi Biotec (7 mentions)
Penicillin, by Eurobio Scientific (6 mentions)
Streptomycin, by Eurobio Scientific (6 mentions)
L glutamine, by Eurobio Scientific (6 mentions)
Sodium pyruvate, by Eurobio Scientific (5 mentions)
Fetal bovine serum (fbs), by Eurobio Scientific (5 mentions)
Hepes buffer, by Eurobio Scientific (3 mentions)
Mycoprobe mycoplasma detection kit, by R&D Systems (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Insulin, by Merck Group (2 mentions)
Collagen 1, by BD (2 mentions)
Hepatocyte attachment medium, by Thermo Fisher Scientific (2 mentions)
Insulin like growth factor 2, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Eurobio Scientific (1 mentions)
27 protocols using rpmi 1640
1
Cell Culture in RPMI-1640 Media
2
Characterizing Murine Tumor Cell Lines
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The murine mammary tumor cell line 4T1 (a gift from Alexzander Asea of Texas A&M Health Science Center College of Medicine, Temple, TX, USA), murine fibroblasts 3T3 cell line (ATCC® CRL-1658™), and HPdLF Human Periodontal Ligament Fibroblasts (Lonza CC-7049) were cultured in RPMI-1640 (Eurobio, Les Ulis Cedex B, France) supplemented with heat-inactivated fetal calf serum (10%) (Eurobio), 2 mM l -glutamine, 100 U/mL penicillin, 100 μg/ml streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37 °C and 5% CO2. Cells were grown until 75% confluence and passaged using trypsin/EDTA 1X (Eurobio), washed with phosphate-buffered saline 1X (PBS), and resuspended in supplemented RPMI-1640. Cells were treated with murine TGFβ (Cell Signaling Technology, Beverly, MA, USA) and RSV (Sigma-Aldrich, St. Louis, MO-USA). P2Et half maximal inhibitory concentration (IC50) for 3T3 (142.7 μg/ml) and 4T1 (34.1 μg/ml) was obtained by MTT assay (Supplementary Fig. 2 ).
3
Murine Melanoma and Breast Cancer Cell Lines
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The murine melanoma B16F10 cell line was kindly provided by PR (Ludwig Center for Cancer Research, Department of Oncology—Faculty of Biology and Medicine University of Lausanne, Switzerland). The B16 mouse model was created in the 1970s from a melanoma that developed spontaneously in the ear of a female C57BL/6 mouse and was then passaged in vivo to create the B16F10 line (21 (link)). The murine breast cancer 4T1 cell line was grown in BALB/c mice and developed into a highly tumorigenic and invasive tumor that can spontaneously metastasize from a primary tumor in the mammary gland to multiple distant sites (22 (link)). The 4T1 mammary tumor cells were kindly provided by Dr Alexzander Asea (Texas A&M Health Science Center College of Medicine, Temple, TX). Cells were cultured in RPMI-1640 (Eurobio, Toulouse, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Eurobio), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M HEPES buffer and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO2. Cells were grown until 75% confluency and passaged using trypsin/1X EDTA (Eurobio), washed with PBS and resuspended in supplemented RPMI-1640.
4
Cultivation of Murine Melanoma Cell Lines
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Melanoma B16F10 and B16OVA cell lines were kindly provided by PR (Ludwig Center for Cancer Research, Department of Oncology—Faculty of Biology and Medicine University of Lausanne, Switzerland). Cells were cultured in RPMI-1640 (Eurobio, Toulouse, France) supplemented with heat-inactivated fetal calf serum (10%) (Eurobio), 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M Hepes buffer and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37 °C and 5% CO2. Cells were grown until 75% confluence and passaged using trypsin/EDTA (ethylene-diamine-tetra-acetic acid) 1 × (Eurobio), washed with PBS and resuspended in supplemented RPMI-1640. Tumor cells were proven Mycoplasma-free using a MycoProbe Mycoplasma Detection Kit (R&D Systems, Minneapolis, MN, USA).
5
Lymphedema and Whole-Body Imaging Protocols
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For both models of lymphedema and whole-body imaging, filariae were injected subcutaneously. Before the injection, larvae were manually counted under a bench stereomicroscope while they were collected with a pipette and then breech-loaded into a U-100 insulin syringe. Despite prior silanization of syringes and needles, approximately 10–20% of larvae stuck to the syringe surface; therefore we manually counted these remaining larvae for the exact determination of inoculated larvae.
Mice with primary thigh lymphedema. 3–7-month old Chy3 mice with palpable lymphedema or their aphenotypic littermates were injected subcutaneously in the shank region of the rear paw with 40 infective L3 larvae in 100 μl of RPMI 1640.
Mice with secondary tail lymphedema. One month after the tail surgery, control mice, sham-operated, and mice with tail lymphedema (tail ⌀ at least double the size of sham-operated mice) were injected subcutaneously with 40 infective L3 larvae in 50 μl of RPMI 1640 (Eurobio, France) distal to the wound on the tail (two-thirds of the tail length measured from its base).
Whole-body imaging. Fifty VivoTag XL 680-stained filaria larvae in 200 μl of Hartmann’s solution were injected subcutaneously at the dorsolateral side of the lumbar region.
Mice with primary thigh lymphedema. 3–7-month old Chy3 mice with palpable lymphedema or their aphenotypic littermates were injected subcutaneously in the shank region of the rear paw with 40 infective L3 larvae in 100 μl of RPMI 1640.
Mice with secondary tail lymphedema. One month after the tail surgery, control mice, sham-operated, and mice with tail lymphedema (tail ⌀ at least double the size of sham-operated mice) were injected subcutaneously with 40 infective L3 larvae in 50 μl of RPMI 1640 (Eurobio, France) distal to the wound on the tail (two-thirds of the tail length measured from its base).
Whole-body imaging. Fifty VivoTag XL 680-stained filaria larvae in 200 μl of Hartmann’s solution were injected subcutaneously at the dorsolateral side of the lumbar region.
6
Culturing and Activating Human ILC2s
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Freshly sorted ILC2s were directly seeded after the FACS-sorting in 200μL of RPMI-1640 (Eurobio) supplemented with 8% of human serum, 1% penicillin-streptomycin 10000U/mL (Gibco), 1% L-Glutamine (Gibco), 1% non-essential amino acids (Gibco), 1% Na Pyruvate (Gibco), kanamycin 100x (Gibco) and 0.1% of 2β-mercaptoethanol 5x10−2M (Sigma), supplemented with 100U/mL recombinant human (rh) IL-2 (PeprtoTech) and 5ng/mL rhIL-7 (PeproTech). When indicated, 3000 mouse stromal OP9 cells (kindly provided by Prof. H. Spits and Dr B. Blum Amsterdam UMC Research Institutes), 1µg/mL PHA (Sigma) or the cytokine cocktail composed of 50ng/mL rhIL-25 (PeproTech), 50ng/mL rhIL-33 (PeproTech) and 50ng/mL rhTSLP (PeproTech) were added. Cells were grown at 37°C under 5% CO2.
7
Mobilization and Isolation of CD34+ HSC
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Example 29
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+-enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
8
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).
9
Mobilization and Expansion of CD34+ Stem Cells
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Example 38
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+-enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
10
Cell Culture of B16-F10 and 4T1 Cells
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B16-F10 and 4T1 cells were cultured in a 37 °C and 5% CO2 incubator with RPMI-1640 (Eurobio, Toulouse, France) with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate [19 (link)]. Abs used for cell surface and intracellular staining were previously described [19 (link)].
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