NB4 human APL cells were a gift from Dr. David Scheinberg (Memorial Sloan Kettering Institute, New York, NY, USA). The ATRA-resistant NB4R cell line was a gift from Prof. Pier Paolo-Pandolfi (Beth Israel Deaconess Cancer Centre, Boston, MA, USA). Cells were cultured at 37°C, 5% CO2 in HyClone RPMI 1640 medium (GE Healthcare Life Sciences SH30027), supplemented with 10% foetal calf serum (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Invitrogen, 10378-016). THP-1 cells (monocytic-lineage) were from the American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 (1x) medium (Gibco by Life technologies, 21870-076), supplemented with 10% foetal calf serum and 1% penicillin/streptomycin. Cells were seeded at 5x104 cells/ml prior to treatment. ATRA (Sigma-Aldrich, R2625) was used to induce differentiation at 1 μM, diluted from a 1 mM stock in 100% ethanol (EtOH). Control populations were treated with 0.1% v/v EtOH. Valproic acid (Sigma-Aldrich, P4543) was used at 1 mM concentration, diluted from a 500mM stock in H20.
Rpmi 1640 1x medium
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
RPMI 1640 (1x) medium is a cell culture medium designed to support the growth and maintenance of a variety of cell types in vitro. It provides essential nutrients, vitamins, and salts required for cellular metabolism and proliferation. The medium is formulated to maintain physiological pH and osmolarity.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hyclone rpmi 1640 medium, by GE Healthcare (1 mentions)
Thp 1 cell, by American Type Culture Collection (1 mentions)
Foetal calf serum, by Thermo Fisher Scientific (1 mentions)
Foetal calf serum, by Merck Group (1 mentions)
Valproic acid, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Phytohemagglutinin a, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Pbmcs, by STEMCELL (1 mentions)
Firefly luciferase lentivirus puromycin, by AMS Biotechnology (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Trypsin edta 1, by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Neubauer chamber, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Egf epidermal growth factor, by Merck Group (1 mentions)
6 protocols using rpmi 1640 1x medium
1
Acute myeloid leukemia cell culture
2
Equine PBMC Stimulation with IL-4 and IL-10
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Horse PBMCs were isolated as per previous protocol [16 ]. Briefly, PBMCs were seeded at a density of 2×105 per well in RPMI 1640-1X medium (GIBCO Life Technologies™, UK) supplemented with 10% fetal calf serum (Sigma, USA), 100 U/ml penicillin, and 100 U/ml streptomycin. Different concentrations of recombinant IL-4 and IL-10 ranging from 250 to 1500 ng/ml (at 250 ng increment) were used for the stimulation of PBMCs. The PBMCs were incubated at 37°C in humid atmosphere of 5% CO2 tension. Culture plates were regularly monitored under microscope till harvesting. Cells were harvested at defined time interval for different assays such as 72 h for lymphocyte proliferation, 48 h for cytokine enzyme-linked immunosorbent assay (ELISA), and 3 h and 24 h for real-time polymerase chain reaction (PCR). Concanavalin A (5 µg/ml) (Sigma, USA) and phytohemagglutinin-A (10 µg/ml) (Sigma, USA) were used as positive control mitogen. PBS-stimulated equine PBMCs were used as negative control. Sham control eluates were used for ascertaining residual proliferative effect of purified fractions obtained during purification process.
3
Ovarian Cancer Cell Lines and CAFs Co-Culture
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The luciferase-tagged SKOV-3 ovarian cancer cell line and the CAFs cell line Human ovarian cancer fibroblast IHFOT-208 were provided by Dr. Michael Birrer (University of Arkansas). The OVCAR-8 cell line was provided by Dr. Geeta Mehta (University of Michigan). The OVCAR-8 cell line was tagged with luciferase using retroviral infection with Firefly Luciferase Lentivirus Puromycin (Amsbio, MA). Luciferase-tagged SKOV-3 and OVAR-8 cells were cultured in RPMI 1640 1X medium, 10% FBS (Gibco; Waltham, MA), and 1% penicillin/streptomycin (ThermoFisher Scientific; Waltham, MA) supplements. IHFOT-208 cells were cultured with DMEM (Gibco, Waltham, MA) 1X with 4.5 g/L glucose, L-glutamine, and sodium pyruvate also supplemented with 10% FBS and 1% penicillin/streptomycin. The CS99 cell line was provided by Gloria Huang (Yale School of Medicine) and was cultured with DMEM (Gibco, Waltham, MA) 1X with 4.5 g/L glucose, L-glutamine, and sodium pyruvate also supplemented with 10% FBS and 1% penicillin/streptomycin. PBMCs were purchased from Stem Cell technologies and were stored in liquid nitrogen until they were used in co-culture with the cancer cells.
4
Cell Culture and miRNA Extraction Protocol
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Initially a cryopreserved vial of each cell line was thawed. The RCC-FG2 and 786-O cell lines were maintained in RPMI 1640 (1X) medium (Gibco®), supplemented with 10% of FBS (Fetal Bovine Serum) (Gibco®) and 1% of Pen-Strep (Gibco®). The HKC-8 cell line was kept in DMEM/F12 medium (Gibco®), supplemented with ITS (Insuline-transferrine-selenium) (Sigma-Aldrich®), Pen-Strep (Gibco®), EGF (Epidermal Growth Factor) (Sigma-Aldrich®), Hepes buffer (Gibco®) and Hydrocortisone (Sigma-Aldrich®). Both cell lines were maintained in a 5% CO2 incubator at 37°C.
When the desired confluence was achieved (80–90%) the medium, in which the cells were being cultured, was collected for miRNA extraction and the cells were trypsinized, using 0.05 % trypsin-EDTA (1×) (Gibco®) and counted using a Neubauer chamber and Tripan-Blue dye (Gibco®). After counting, approximately two million cells were centrifuged to form a pellet for miRNA extraction and the remaining cells were kept in culture. This procedure was repeated five times for each cell line.
When the desired confluence was achieved (80–90%) the medium, in which the cells were being cultured, was collected for miRNA extraction and the cells were trypsinized, using 0.05 % trypsin-EDTA (1×) (Gibco®) and counted using a Neubauer chamber and Tripan-Blue dye (Gibco®). After counting, approximately two million cells were centrifuged to form a pellet for miRNA extraction and the remaining cells were kept in culture. This procedure was repeated five times for each cell line.
5
Cell Culture Conditions for Diverse Cell Lines
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The CCF-STTG1 astrocyte cell line (The European Collection of Authenticated Cell Cultures (ECACC)) and the newly developed primary immortalised human astrocyte HASTR/ci35 cell line were cultured in RPMI Medium 1640 (1X) (Gibco, Waltham, MA, USA) containing 10% FBS,100 I.U./mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and 2.5 ug/mL Plasmocin (InvivoGen, San Diego, CA, USA). Vero cells and primary immortalised human astrocytes, foetal-hTERT (abmGood, Richmond, BC, Canada) were cultured in Dulbecco’s Modified Eagle Medium (Gibco, Waltham, MA, USA) containing 10% foetal bovine serum (FBS), 100 I.U./mL penicillin and 100 μg/mL streptomycin and 2.5 µg/mL Plasmocin. C6/36 cells were cultured using Minimum Essential Medium Eagle (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS and 100 I.U./mL penicillin and 100 μg/mL streptomycin. The Mouse hybridoma B lymphocyte cells were purchased from ATCC and used to produce flavivirus group antigen Antibody (D1-4G2-4-15 (4G2)). They were cultured using Hybri-Care Medium, containing 10% FBS, 1.5 g/L sodium bicarbonate and 100 I.U./mL penicillin and 100 μg/mL streptomycin. All the cells were cultured by incubating at 37 °C and 5% CO2 conditions, except the C6/36 cell line, which was incubated at 28 °C with no CO2.
6
Evaluating B. pertussis Infection in Cord Blood
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Fresh heparinized cord blood instead of whole blood from adult donor was used to mimic an infection in early infancy. Cord blood samples were provided by the biocollection of the Cell J o u r n a l P r e -p r o o f Therapy Unit from the Biological Resources Center of Saint-Louis Hospital (Paris, France). 200 L of whole cord blood was added to each well of a flat bottom 48-well plates and incubated with 200 L of bacterial suspension diluted in RPMI medium (RPMI Medium 1640 (1X) + GlutaMAXTM-I, Gibco) to the desired concentration, to reach a Multiplicity of infection (MOI, ratio bacteria/cell) of approximately 10. The plates were then incubated at 37°C and 5% CO2 for 4 h (for bacterial survival assays) or 22 h (for cytokine assays). For survival assays, after 4 h of whole cord blood infection, 50 μl of the suspension were serial diluted and plated on BGA to estimate B. pertussis survival. Survival was expressed as a percentage of the initial B. pertussis load. Anti-PT IgG antibody concentrations were measured in all cord blood samples (they were originated from 9 different donors) used in this study (kit Savyon SeroPertussis TM Toxin IgG 1231-01D); all of them had IgG titers < 40 UI/ml and were therefore considered seronegative.
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