The p38α-deficient p38 –/– EV and p38α-reconstituted p38 –/– p38 MEFs were a kind gift from Prof. Matthias Gaestel (Hannover Medical School, Germany). The cells were cultivated in Dulbecco’s modified essential medium (Sigma-Aldrich Chemie GmbH, Munich, Germany) with 5% fetal bovine serum (PAN Biotech GmbH, Aidenbach, Germany), 1% penicillin, 1% streptomycin (PAA Laboratories GmbH, Pasching, Austria), 1 mM sodium pyruvate (Biochrom AG, Berlin, Germany) and 1 mM MEM None-essential amino acids (Gibco, Life Technologies, Paisley, UK) at 37°C and 5% CO2. When the cells reached confluence, they were passaged.
Dulbecco s modified essential medium
Manufactured by Merck Group
Sourced in Germany
Dulbecco's modified essential medium (DMEM) is a cell culture medium that provides essential nutrients, amino acids, vitamins, and other components to support the growth and maintenance of various cell types in vitro. It is a widely used and versatile medium that is suitable for a range of cell lines, including adherent and suspension cultures.
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Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Sodium pyruvate, by Harvard Bioscience (1 mentions)
Penicillin, by GE Healthcare (1 mentions)
Streptomycin, by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
H9c2 cell, by American Type Culture Collection (1 mentions)
Penicillin, by Corning (1 mentions)
Penicillin streptomycin, by Leibniz Institute DSMZ (1 mentions)
Mycoalert assay control set, by Lonza (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Chlorpromazine, by Merck Group (1 mentions)
Odn2006, by InvivoGen (1 mentions)
Cl264, by InvivoGen (1 mentions)
Ammonium iron 3 citrate, by Merck Group (1 mentions)
Methyl β cyclodextrin, by Merck Group (1 mentions)
5 protocols using dulbecco s modified essential medium
1
Cultivation of p38α-deficient and reconstituted MEFs
2
NHDF Cell Culture and PHNQs Treatment
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Normal human dermal fibroblasts (NHDF) were grown in Dulbecco’s Modified Essential Medium (DMEM; Sigma Merck, Darmstadt, Germany) high glucose (4.5 g L−1), supplemented with 20% (w/v) fetal bovine serum, 2 mM L-glutamine, 100 U mL−1 penicillin, and 100 mg mL−1 streptomycin (Sigma Merck, Darmstadt, Germany). Cells were cultured in a humidified 5% (v/v) CO2 atmosphere at 37 °C. Cell culture medium was changed every 2–3 days, and cells were used at passage 5–10. For the experiments, the cells were seeded in multiwell plates and grown to sub-confluence (80%) before the PHNQs treatment.
The lyophilized PHNQs extract was reconstituted in Hybri-Max sterile-filtered dimethyl sulfoxide (DMSO; Sigma Merck, Darmstadt, Germany) immediately before its use (stock solution, 10 mg mL−1, kept in the dark to avoid any possible photodegradation) and dissolved in culture medium. NHDF cells were incubated with the indicated concentrations of PHNQs extract for 24 h (see below). The final concentration of DMSO in culture medium never exceeded 1% (v/v).
The lyophilized PHNQs extract was reconstituted in Hybri-Max sterile-filtered dimethyl sulfoxide (DMSO; Sigma Merck, Darmstadt, Germany) immediately before its use (stock solution, 10 mg mL−1, kept in the dark to avoid any possible photodegradation) and dissolved in culture medium. NHDF cells were incubated with the indicated concentrations of PHNQs extract for 24 h (see below). The final concentration of DMSO in culture medium never exceeded 1% (v/v).
3
Culturing Rat Cardiomyoblast H9c2 Cells
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The rat cardiomyoblast H9c2 cells (rat embryonic cardiac myoblasts; ATCC CRL-1446, VA, USA) were grown in low glucose Dulbecco’s modified essential medium (Sigma-Aldrich D6046, MO, USA) with 10% HyClone™ Cosmic Calf™ Serum (U.S.) and 1% penicillin (Corning®, 30-002-CI), and incubated in a humidified incubator with 5% CO2 at 37 °C.
4
Culturing Breast Cancer Cell Lines
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BT-474 (ATCC, HTB-20) and brain seeking BT-474-Br (generously provided by Dihua Yu (MD Anderson Cancer Center)) cells were grown in RPMI-1640 (Sigma-Aldrich, R5886) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, F7524), 1% vol/vol penicillin/streptomycin (Sigma-Aldrich, P0781-100ML) and L-glutamine. MDA-MB-361 cells were grown in Dulbecco’s modified essential medium (DMEM; Sigma-Aldrich, D5769) supplemented with 20% FBS, 1% vol/vol penicillin/streptomycin and L-glutamine. Telomerase immortalized foreskin fibroblasts (TIFF, generously provided by J. Norman (Beatson Institute for Cancer Research)) and JIMT-1 (DSMZ, ACC 589) cells were grown in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin and L-glutamine. All cells were cultured in a humidified incubator set at 5% CO2 and 37 °C. All cells were tested bimonthly – every 2 months – to ensure mycoplasma-free cell culture using MycoAlertTM mycoplasma detection kit (Lonza, #LT07-418) and MycoAlertTM assay control set (#LT07-518). Cell lines were not separately authenticated within this study. The antibodies used are described in Supplementary Table 1 . Previously published plasmids used in this study are summarized in Supplementary Table 2 .
5
Characterization of Iron Nanomedicine Reagents
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4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), adenosine triphosphate (ATP), ammonium iron (III) citrate, propidium iodide, chlorpromazine, methyl-beta-cyclodextrin, transferrin, Dulbecco's Phosphate Buffered Saline (PBS) without calcium chloride and magnesium chloride, PBS with calcium chloride and magnesium chloride, fetal bovine serum (FBS), trypsin, ethylenediaminetetraacetic acid (EDTA), RPMI-1640 containing both 20 mM HEPES, L-glutamine and sodium bicarbonate, Dulbecco's Modified Essential Medium containing 4.5 g/L glucose and L- glutamine, and antibioticÀantimycotic solution containing pen-icillinÀstreptomycin and antimycotics were all obtained from Sigma-Aldrich (Zwijndrecht, the Netherlands). Ficoll-Paque Plus was obtained from Fisher Scientific (Landsmeer, the Netherlands). Normocin, blasticidin, zeocin, HEk-Blue Selection, PAM3SCK, Poly(I:C) LPS-EK, CL264, ODN2006, and QUANTI-Blue were obtained from Invivogen (Toulouse, France). Ferric gluconate, iron sucrose, ferric carboxymaltose and iron isomaltoside 1000 were kindly provided by Vifor Pharma (Glattbrug, Switzerland) and freshly used before the expiration date. All iron nanomedicines were endotoxin free, determined by the Pyrogene Recombinant Factor C assay (Lonza Benelux bv, Breda, the Netherlands).
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