MCF7, MDA-MB231, HCT116, HeLa and HEK293T (from ATCC, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (BioWest, Nuaillé, France) as described35 (link). MCF10A cells were grown in DMEM/F12 medium (BioWest) with L-glutamine and HEPES, supplemented with 5% (v/v) horse serum, 100 μg/ml streptomycin and 100 U/ml penicillin from Gibco (Thermo Fisher Scientific, Waltham, MA, USA), and 20 ng/ml epidermal growth factor, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera enterotoxin and 10 μg/ml insulin from Sigma-Aldrich (St. Louis, MO, USA). hTERT RPE1 cells were grown in DMEM/F12 medium (BioWest) with L-glutamine and HEPES, supplemented with 5% (v/v) fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin from Gibco. NP40 extracts were prepared at 4 °C in 150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1% Nonidet P-40 (NP40), 10% glycerol, 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin and 10 μg/ml chymostatin for 20 min. Extracts were centrifuged at 20,000 g for 20 min and supernatants frozen in liquid nitrogen and stored at −80 °C. Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA).
Dmem f12 medium
Manufactured by Biowest
Sourced in France, United States
DMEM-F12 medium is a basal medium formulation commonly used for the in vitro culture of a variety of cell types, including mammalian cells. It provides a balanced salt solution, amino acids, vitamins, and other essential nutrients required for cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Insulin, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Biowest (3 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Amphotericin b, by Biowest (2 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Cholera enterotoxin, by Merck Group (1 mentions)
Minimum essential medium (mem), by Corning (1 mentions)
L glutamine, by Corning (1 mentions)
Sh sy5y atcc crl 2266, by American Type Culture Collection (1 mentions)
Penicillin streptomycin mixture, by Corning (1 mentions)
Dulbecco s modified, by Biowest (1 mentions)
9 protocols using dmem f12 medium
1
Cell Culture and Protein Extraction Protocol
2
Virus Infection Assay in Mammalian Cell Lines
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Virus infections were performed on Vero ATCC CCL-81 or SH-SY5Y ATCC CRL-2266 cells (ATCC, Manassas, VA). Vero cells were cultured in minimal essential medium (MEM) (Corning, Manassas, VA) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Paisley, UK), and SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium (Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum. A penicillin-streptomycin mixture (Corning) and l -glutamine (Corning) were also added to cell cultures. WNV New York 99 (60 (link)), USUV SAAR 1776 (60 (link)), American ZIKV PA259459 (61 (link)), a cell-passaged derivative of DENV-2 16681 (16 (link)), VSV Indiana (61 (link)), and CVB5 Faulkner (62 (link)) were used. Procedures for infections in liquid medium and virus titrations in Vero cells in semisolid agar medium were previously described (62 (link), 63 (link)). WNV, USUV, ZIKV, VSV, and CVB5 titers were determined at 24 h postinfection (p.i.). DENV-2 titers were determined at 48 h p.i. A multiplicity of infection (MOI) of 1 PFU/cell was used for all experiments unless otherwise indicated.
3
Breast Cancer Cell Culture Protocols
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Experimental cultures of breast (MDA-MB-231,
MCF-7, and MCF-10A) were obtained from the American Type Culture Collection
(ATCC) and cultured in tissue culture dishes (Corning, NY) at 37 °C
in 5% CO2 atmosphere. Phosphate-buffered saline was obtained
from Gibco. Experiments were performed on cells within 20 passages.
The breast cancer cells MDA-MB-231 and MCF-7 cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM; Biowest
L0106, France) supplemented with 10% FBS (Gibco, NY), 1%l -glutamine (PAA Laboratories, Austria), and 1% penicillin/streptomycin
(HyClone, UT). The MCF-10A cells were maintained in a DMEM-F12 medium
(Biowest L0093, France) supplemented with 7.5% FBS (Gibco, NY), epidermal
growth factor (Invitrogen), insulin (Sigma-Aldrich), hydrocortisone
(Sigma-Aldrich) and 0.4% gentamicin (Gibco, NY), and 1% penicillin/streptomycin
(HyClone, UT). Bright-field images of the adhered cells were taken
using a Nikon Eclipse TS100 inverted microscope, with a 10× objective
and Nikon digital Sight DS-L3 camera.
MCF-7, and MCF-10A) were obtained from the American Type Culture Collection
(ATCC) and cultured in tissue culture dishes (Corning, NY) at 37 °C
in 5% CO2 atmosphere. Phosphate-buffered saline was obtained
from Gibco. Experiments were performed on cells within 20 passages.
The breast cancer cells MDA-MB-231 and MCF-7 cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM; Biowest
L0106, France) supplemented with 10% FBS (Gibco, NY), 1%
(HyClone, UT). The MCF-10A cells were maintained in a DMEM-F12 medium
(Biowest L0093, France) supplemented with 7.5% FBS (Gibco, NY), epidermal
growth factor (Invitrogen), insulin (Sigma-Aldrich), hydrocortisone
(Sigma-Aldrich) and 0.4% gentamicin (Gibco, NY), and 1% penicillin/streptomycin
(HyClone, UT). Bright-field images of the adhered cells were taken
using a Nikon Eclipse TS100 inverted microscope, with a 10× objective
and Nikon digital Sight DS-L3 camera.
4
Extracellular Vesicle Differentiation of ATDC-5 Cells
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ATDC-5 cells (25,000 cells/well) were seeded in 48 well plates with DMEM-F12 medium (Biowest) supplemented with 5% FBS Premium (Biowest), 1% penicillin-streptomycin, supplemented with 10 µg/mL transferrin (Sigma-Aldrich) and 5 ng/mL sodium selenite (Sigma-Aldrich). When cells reached confluence 10 µg/mL insulin (Sigma-Aldrich) were added to the medium to induce cell differentiation. Medium was refreshed every other day.
At day 15, cells were incubated with DMEM-F12 medium supplemented with 5% EV-depleted FBS Premium, 1% penicillin-streptomycin, 10 µg/mL transferrin and 5 ng/mL sodium selenite, and treated for 48 h either with one dose of UC (5 µg protein/1.51 × 107 particles), UCw/o (5 µg protein /1.37 × 109 particles), EV (1.37 × 109 particles), EVw/o (1.37 × 109 particles), Prot (5 µg protein) and Protw/o (5 µg protein). Two independent experiments were performed using 6 replicates in each study.
At day 15, cells were incubated with DMEM-F12 medium supplemented with 5% EV-depleted FBS Premium, 1% penicillin-streptomycin, 10 µg/mL transferrin and 5 ng/mL sodium selenite, and treated for 48 h either with one dose of UC (5 µg protein/1.51 × 107 particles), UCw/o (5 µg protein /1.37 × 109 particles), EV (1.37 × 109 particles), EVw/o (1.37 × 109 particles), Prot (5 µg protein) and Protw/o (5 µg protein). Two independent experiments were performed using 6 replicates in each study.
5
Cytotoxicity and Apoptosis Assays
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Minimum Essential Medium (MEM) was purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany), while DMEM/F12 medium, penicillin-streptomycin solution, amphotericin B solution, L-glutamine and trypsin were purchased from Biowest (Nuaillé, France). Fetal bovine serum (FBS), non-essential amino acids, DMSO, Thiazolyl blue tetrazolium bromide (MTT) and JC-1 were obtained from Sigma-Aldrich Chemie GmbH. Dihydroethidium (DHE), dihydrorhodamine 123 (DHR), fluorescein-di-β-D-galacto-pyranoside (FDG) and hoechst 33342 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Annexin-V-FITC (AV) and propidium iodide (PI) Apoptosis Detection Kit was purchased from Abcam (Cambridge, UK). Bovine serum albumin (BSA) was from Serva (Heidelberg, Germany) and Triton™ X-100 was from Merck KGaA (Darmstadt, Germany). Anti-phospho-histone H2A.X (Ser139) rabbit primary antibody was from Cell Signaling Technology® (Danvers, MA, USA) and Alexa Fluor® 488 goat anti-rabbit IgG (H + L) secondary antibody was purchased from Thermo Fisher Scientific.
6
Cultivation of Neuroblastoma SH-SY5Y Cells
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The neuroblastoma cell line (SH-SY5Y) was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) Bank (ACC 209). Cells were cultivated at 37 °C and 5% CO2 with DMEM:F12 medium containing 1% antibiotic/antimycotic (amphotericin B, penicillin, and streptomycin) (Biowest, Nuaillé, France) and 10% (v/v) fetal bovine serum (FBS) (Biowest, Riverside, MO, USA).
7
SH-SY5Y Cell Culture for Bioactivity
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The biological activities of fractions were conducted on an in vitro cellular model of human neuroblastoma (SH-SY5Y cells, strain number ACC 209) previously acquired from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) biobank. The cells were cultured according to the supplier’s instructions. SH-SY5Y cells grew in DMEM:F12 medium (Biowest, Riverside, MO, USA) supplemented with 10% (v/v) fetal bovine serum (Biowest, Riverside, MO, USA) and 1% of antibiotic mixture constituted by 1% penicillin/streptomycin (Sigma, Rehovot, Israel). SH-SY5Y cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C.
8
Renal Cell Carcinoma Cell Lines: Cultivation and Knockdown
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The human renal clear cell carcinoma cell line Caki-1 and the Xp11.2 translocation renal cell carcinoma cell lines (UOK109, UOK120, UOK124, UOK145, and UOK146) were maintained in DMEM (Cytiva, Marlborough, MA). The human renal proximal tubular epithelial cell line HK-2 was maintained in DMEM/F-12 medium (Biowest, Bradenton, FL). All media were supplemented with 10% FBS (Cytiva, Marlborough, MA) and 1% penicillin streptomycin (Thermo Fisher Scientific, Waltham, MA). All cells were incubated at 37 °C in a humidified atmosphere containing 5% CO 2 . Transfection was performed with short interfering RNAs (siRNAs) against TFE3 and PPARGC1A (Bioneer, Daejeon, Korea) using Lipofectamine RNAiMAX transfection reagents (Thermo Fisher, Waltham, MA) following the manufacturer's instructions.
9
Endometrial Protein Extraction and Analysis
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The extraction and analysis of the proteins from the endometrial biopsies were performed as previously described by our group [42 (link)]. Briefly, endometrial biopsy samples were collected and placed in a DMEM-F12 medium supplemented with HEPES, penicillin/streptomycin, and amphotericin B (all Biowest, Nuaille, France). Protein lysates from endometrial tissue were prepared and processed for mass spectrometric analysis (Thermo Scientific, Dreieich, Germany). Proteins and peptides were identified and quantified, and data analysis was performed within the MaxQuant Software (version 1.5.7.0, MPI for Biochemistry, Planegg, Germany) [61 (link)].
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