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5 protocols using cell culture flasks and dishes

1

Cell Culture Protocol for Mycotoxin Assays

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Cell culture media and supplements were purchased from GIBCO Invitrogen (Karlsruhe, Germany), cell culture flasks and dishes from Sarstedt (Nürnbrecht, Germany) and from Ibidi (Ibidi GmbH, Martinsried, Germany). 17β-Estradiol (E2), alternariol (AOH), quercetin (QUE) and Triton-X 100 were purchased from Sigma-Aldrich (Schnelldorf, Germany). CK2α1 Kinase Enzyme System, ADP-Glo Assay as well as Maxwell 16 LEV simplyRNA Cell Extraction kits were obtained from Promega (Mannheim, Germany). QuantiTect® Reverse Transcription kit, QuantiTect® SYBR® Green PCR kit and primers for ESR1, ESR2, ALPP, GADPH and ACTB were purchased from Quiagen (Hilden, Germany). ATX-II was isolated from an extract of Alternaria alternata cultured on rice as previously described (Puntscher et al. 2018a (link)).
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2

Cultivation of HT-29 Colon Cancer Cells

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HT-29 colon carcinoma cells were acquired from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% (v/v) of heat-inactivated fetal calf serum and 1% (v/v) of penicillin/streptomycin. Dulbecco’s phosphate-buffered saline (DPBS), cell culture media, and supplements were obtained from GIBCO Invitrogen (Karlsruhe, Germany); cell culture flasks and dishes were purchased from Sarstedt (Nümbrecht, Germany). Cells were grown to 70–90% confluence at 37 °C in humidified conditions in 5% CO2 atmosphere.
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3

Cellular Responses to Environmental Toxins

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Cell culture media and supplements were obtained from GIBCO Invitrogen (Karlsruhe, Germany), cell culture flasks and dishes from Sarstedt (Nürnbrecht, Germany). 17β-Estradiol, alternariol, alternariol monomethyl ether, ethidium bromide, neutral red, triton X-100, benzo[a] pyrene and 4-nitrophenyl phosphate were purchased from Sigma-Aldrich (Schnelldorf, Germany). Maxwell 16 LEV simplyRNA kit was obtained from Promega, QuantiTect ® Reverse Transcription Kit, QuantiTect ® SYBR ® Green PCR Kit and primers for CYP1A1, ESR1, ESR2, ALPP, GADPH and ACTB from Qiagen (Hilden, Germany). Normal and low melting Agarose were purchased from Bio-Rad, formamidopyrimidine-DNA glycosylase from New England Biolabs (Frankfurt, Germany).
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4

Cr2O3 Particles Synthesis and Evaluation

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The Cr2O3 particles were purchased from Nanostructured & Amorphous (Katy, TX, USA) Lot: 1910-091918 (particle A), and Sigma Aldrich (Steinheim, Germany) Lot: 634239 (particle B). Particle C (Lot: CHC 2018-19) was kindly provided by Lanxess (Cologne, Germany). CrCl3 hexahydrate (≥97%) and K2Cr2O7 (≥99.5%) were purchased from Carl Roth (Karlsruhe, Germany).
Dimethyl sulfoxide (≥99.9%) and 1,5-diphenylcarbazide (≥97.0%) were purchased from Sigma Aldrich (Steinheim, Germany). A CellTiter-Glo® Luminescent Cell Viability Assay was purchased from Promega (Madison, WI, USA). All PCR consumables, including PCR tubes, strips, reaction tubes, and tubules, as well as cell culture dishes and flasks, were obtained from Sarstedt (Nuembrecht, Germany). The primer pairs were synthesized by Eurofins Genomics (Ebersberg, Germany) or Fluidigm (San Francisco, CA, USA). The DNA suspension buffer, PCR-certified water, and TE buffer were obtained from Teknova (Hollister, CA, USA). The 2X Assay Loading Reagent and 20X DNA Binding Dye Sample Loading Reagent were purchased from Fluidigm (San Francisco, CA, USA). Bio-Rad (Munich, Germany) provided the 2X SsoFastTM EvaGreen® Supermix with Low ROX and the 2X SYBR Green Supermix. The 2X TaqMan® PreAmp Master Mix was obtained from Applied Biosystems (Darmstadt, Germany) and exonuclease I from New England Biolabs (Frankfurt am Main, Germany).
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5

Characterization of CuO Nanoparticles

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All chemicals (p.a. grade), cell culture medium, and supplements were obtained either from Carl Roth GmbH (Karlsruhe, Germany) or Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) with the exception of fetal bovine serum (FBS), which was bought at Thermo Fisher Scientific GmbH (Dreieich, Germany). Cell culture dishes and flasks, reaction tubes, PCR tubes, and other consumables were purchased from Sarstedt (Nuembrecht, Germany). CuO NP were kindly provided by Dr. Wendel Wohlleben (BASF SE, Germany) in the course of the BMBF-funded project MetalSafety and originally synthesized and obtained from by PlasmaChem (Berlin, Germany). This particle species was also previously examined in the EU FP7-Project SUN, including oral and inhalation in vivo studies [28 (link),60 (link)]. Primer pairs were synthesized by Eurofins (Ebersberg, Germany). DNA suspension buffer, PCR certified water, and TE buffer were obtained from Teknova (Hollister, USA). The 2× Assay Loading Reagent, 20× DNA Binding Dye, and IFCs were purchased from Fluidigm (San Francisco, USA). The 2× SsoFast EvaGreen Supermix was provided by Bio-Rad (Munich, Germany), and the 2× TaqMan PreAmp Master Mix was bought from Applied Biosystems (Darmstadt, Germany). Exonuclease I was obtained from New England Biolabs (Frankfurt am Main, Germany).
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