Human HNSCC cell lines FaDu, Cal27, SCC4, SCC9 and SCC25 were purchased from ATCC (https://www.lgcstandards-atcc.org ). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (Invitrogen, Germany), 2 mM l -glutamine and 50 μg/ml penicillin-streptomycin in humidified and sterile conditions with 6% CO2 at 37 °C. Cell cultures were regularly screened to exclude mycoplasma contamination (Venor®GeM Classic Mycoplasma Detection Kit, Minerva Biolabs) according to manufacturer’s recommendation, and the authentication of all cell lines was confirmed by the Multiplex Human Cell Line Authentication Test (Multiplexion, Germany, latest update April 2019).
Venor gem classic mycoplasma detection kit
Manufactured by Minerva Biolabs
Sourced in Germany
The Venor GeM Classic Mycoplasma Detection Kit is a PCR-based test kit designed to detect the presence of mycoplasma contamination in cell cultures. The kit includes all necessary reagents and controls to perform the analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Te 15, by RIKEN BioResource Center (2 mentions)
Te 10, by RIKEN BioResource Center (2 mentions)
Te 11, by RIKEN BioResource Center (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Monomac6, by Leibniz Institute DSMZ (2 mentions)
Thp 1, by Leibniz Institute DSMZ (2 mentions)
Scc25, by American Type Culture Collection (2 mentions)
Cal27, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Scc 4, by American Type Culture Collection (1 mentions)
Ficoll, by Merck Group (1 mentions)
Decitabine dac, by Merck Group (1 mentions)
22 protocols using venor gem classic mycoplasma detection kit
1
Establishment and Authentication of HNSCC Cell Lines
2
Isolation and Culture of CML Cells
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Mononuclear cells were isolated from peripheral blood (PB) or BM of 31 samples (17 CP-CML, 5 AP-CML, 9 BC-CML) from 23 CML patients using Ficoll (Sigma, St Louis, MO, USA). PB and BM samples were collected from all patients at diagnosis and from a subset of four patients during their follow-up. All four patients progressed to AP or BC during therapy with BCR-ABL1 tyrosine kinase inhibitors. Clinico-pathological characteristics of the patients are shown in Supplementary Table S1 . Mononuclear cells obtained from five individuals were used as controls. All donors gave written informed consent. The study was approved by the ethics committee of the Medical University of Vienna, Austria (no. 1594/2015).
Authenticated cell lines K562, KCL22, NALM-1, TOM-1 and BV-173 were purchased from the Leibniz Institute DSMZ (Braunschweig, Germany). KU812 cells were kindly provided by Dr K Kishi (Niigata University, Niigata, Japan), and K562 R cells (imatinib-resistant) were kindly provided by James D Griffin (Dana Farber Cancer Institute, Boston, MA, USA). Cells were cultured in RPMI1640 medium with 10% fetal calf serum at 37 °C. Mycoplasma contamination was tested using the Venor GeM Classic Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany). Aza-dC and TSA treatment was performed as reported.21 (link) Untreated control cells were cultured in parallel.
Authenticated cell lines K562, KCL22, NALM-1, TOM-1 and BV-173 were purchased from the Leibniz Institute DSMZ (Braunschweig, Germany). KU812 cells were kindly provided by Dr K Kishi (Niigata University, Niigata, Japan), and K562 R cells (imatinib-resistant) were kindly provided by James D Griffin (Dana Farber Cancer Institute, Boston, MA, USA). Cells were cultured in RPMI1640 medium with 10% fetal calf serum at 37 °C. Mycoplasma contamination was tested using the Venor GeM Classic Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany). Aza-dC and TSA treatment was performed as reported.21 (link) Untreated control cells were cultured in parallel.
3
Melanoma Spheroid Generation and Isolation
4
Decitabine Treatment of Head and Neck Cancer Cells
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FaDu (RRID:CVCL_1218), Cal27 (RRID:CVCL_1107), SCC4 (RRID:CVCL_1684), SCC9 (RRID:CVCL_1685) and SCC25 (RRID:CVCL_1682) cells were purchased from the American Type Culture Collection (ATCC, https://www.lgcstandards-atcc.org/ (accessed on 21 November 2021) Manassas, VA, USA), and Detroit562 were purchased from Cell Lines Service (CLS, Eppelheim, Germany). All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies, Darmstadt, Germany) and supplemented with 10% fetal bovine serum (Invitrogen, Karlsruhe, Germany), 2 mM L-glutamine and 50 μg/mL penicillin-streptomycin (Sigma-Aldrich, Germany) in humidified and sterile conditions with 6% CO2 at 37 °C. Cell cultures were regularly screened to exclude mycoplasma contamination (Venor®GeM Classic Mycoplasma Detection Kit, Minerva Biolabs, Berlin, Germany) according to manufacturer’s recommendation. Authentication of all cell lines was confirmed by the Multiplex Human Cell Line Authentication Test (Multiplexion, Heidelberg, Germany, latest update 8 March 2022).
Cancer cells (1 × 106 per dish) were seeded in a 10 cm TC dishes (day 0) and were treated with 0.5–1 µM Decitabine (DAC, Sigma-Aldrich, Germany) or DMSO as control for 2 days (day 1–2). On day 3, cells were harvested and further processed for total RNA extraction.
Cancer cells (1 × 106 per dish) were seeded in a 10 cm TC dishes (day 0) and were treated with 0.5–1 µM Decitabine (DAC, Sigma-Aldrich, Germany) or DMSO as control for 2 days (day 1–2). On day 3, cells were harvested and further processed for total RNA extraction.
5
Embryonic Stem Cell Culture Conditions
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ES cells were grown in two different media, serum and 2i, defined as follow. Serum: Glasgow medium (Sigma), 15% FBS (Gibco), 2 mM L-Glutamine, 0.1 mM MEM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM β-mercaptoethanol, 1000 U/mL leukemia inhibitory factor (LIF, Miltenyi); 2i: 50% neurobasal medium (Gibco), 50% DMEM/F12 (Gibco), 2 mM L-Glutamine (Gibco), 0.1 mM β-mercaptoethanol, Ndiff Neuro2 supplement (Milipore), B27 serum-free supplement (Gibco), 1000 U/mL LIF, 3 μM Gsk3 inhibitor CT-99021, 1 μM MEK inhibitor PD0325901. Vitamin C (Sigma) was added at a concentration of 100 ug/mL (Blaschke et al., 2013 (link)).
When in serum, J1, Dnmt-tKO, E14, and all CRISPR-generated mutant ES cells were grown in feeder-free conditions on gelatin-coated plates. TT2, Ehmt2-KO were cultured on a monolayer of mitomycin C-treated mouse embryonic fibroblasts. ES cells were passaged with TrypLE Express Enzyme (Life Technologies, Carlsbad, CA). All 2i ES cells were grown in gelatin-coated plates and passaged every two or three days with Accutase (Life Technologies).
Trichostatin A was added for 24 hr at concentration of 25 or 50 ng/mL
Mycoplasma-free status was assessed using VenorGeM Classic mycoplasma detection kit (Minerva Biolabs).
When in serum, J1, Dnmt-tKO, E14, and all CRISPR-generated mutant ES cells were grown in feeder-free conditions on gelatin-coated plates. TT2, Ehmt2-KO were cultured on a monolayer of mitomycin C-treated mouse embryonic fibroblasts. ES cells were passaged with TrypLE Express Enzyme (Life Technologies, Carlsbad, CA). All 2i ES cells were grown in gelatin-coated plates and passaged every two or three days with Accutase (Life Technologies).
Trichostatin A was added for 24 hr at concentration of 25 or 50 ng/mL
Mycoplasma-free status was assessed using VenorGeM Classic mycoplasma detection kit (Minerva Biolabs).
6
Establishment and Characterization of GBM Cell Lines
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Human GBM cell lines A172, U-87 MG, and LN-18 were obtained from ATCC. Cells were cultivated in DMEM (Gibco) containing 10% FBS (Gibco), 2 mM Glutamine (Gibco), 1 mM Sodium Pyruvate (Gibco), 100 μg/ml Streptomycin, and 100 U/ml Penicillin (Gibco). Cells were authenticated by Multiplex Cell Authentication service (Multiplexion GmbH) and were routinely monitored using the Venor® GeM Classic mycoplasma detection kit (Minerva Biolabs). Twenty-four hours after cell seeding a medium change was done following which, various treatments were carried out. Unless stated otherwise, cells were normally cultivated at normoxic conditions i.e., 18.6% O2 concentration (conc.) (25 (link)) in a SANYO MCO-18AIC incubator with 5% CO2 and at 37°C.
7
Obtaining Cellular Materials for AML Research
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PB and BM samples from patients with AML were obtained upon receipt of their informed consent and in accordance with the Declaration of Helsinki (Ethics Committee approval 219_14B). Mononuclear cells from the PB or BM were obtained with Ficoll-Paque (GE Healthcare, Chicago, IL). Patient characteristics can be found in supplemental Table 1 . MSCs were isolated from iliac crest BM aspirates taken from patients with newly diagnosed AML (Ethics Committee Approval 4777) by density gradient centrifugation and were expanded as previously detailed, while fulfilling uniformly minimal criteria.24 (link)
The human BM stroma cell line HS-5 was purchased from ATCC (Manassas, VA). The AML cell lines OCI-AML3 and MOLM-13 were purchased from DSMZ (Braunschweig, Germany). Mycoplasma contamination was regularly assessed with the Venor GeM Classic Mycoplasma detection kit (Minerva Biolabs, Berlin, Germany), according to the manufacturer’s instructions, and authentication of the cell lines was performed.
The human BM stroma cell line HS-5 was purchased from ATCC (Manassas, VA). The AML cell lines OCI-AML3 and MOLM-13 were purchased from DSMZ (Braunschweig, Germany). Mycoplasma contamination was regularly assessed with the Venor GeM Classic Mycoplasma detection kit (Minerva Biolabs, Berlin, Germany), according to the manufacturer’s instructions, and authentication of the cell lines was performed.
8
Melanoma Cell Lines and Drug Assays
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Melanoma cell lines used in this study were purchased from ATCC and the Leibnitz Institute Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). The SK-HI-SETDB1 cell line is a subclone generated by the parental SKMEL28 cell line, as previously described,13 (link) characterized by high mean SETDB1 amplification. Identity of melanoma cells was authenticated by a cell-line authentication test. Cells were routinely tested for mycoplasma contamination with the VenorGeM Classic mycoplasma detection kit (Minerva Biolabs). Melanoma cells, NHMs, and dermal fibroblasts (isolated from a healthy patient’s foreskin) were cultured in mouse embryonic fibroblast (MEF) medium, composed of DMEM medium (Life Technologies), 10% heat-inactivated fetal calf serum (FCS; Biochrom), 0.1 mM β-mercaptoethanol (Life Technologies), 1% nonessential amino acids (Sigma-Aldrich), and 1% penicillin/streptomycin (Sigma-Aldrich), and stably kept at 37°C and 5% CO2 in a humidified incubator. Drug-based assays were performed, exposing cultured cells to DMSO-dissolved mit (BioTrend), EC-8042 (under development at EntreChem SL), vemurafenib (Selleckchem), or trametinib (Selleckchem), at defined concentrations (final DMSO concentration in cell culture medium < 0.1%).
9
Cultivation of porcine intestinal epithelial cells
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Intestinal porcine epithelial cells (IPEC-J2; ACC 701, Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Culture, Braunschweig, Germany) were cultivated and routinely maintained using DMEM/F12 (1:1) without L-glutamine (Pan Biotech, Aidenbach, Germany), adjusted to 2.4 g/L NaHCO3 (Pan Biotech) and supplemented with 1% insulin–transferrin–selenium ITS (Gibco/Life Technologies, Thermo Fisher Scientific, Vienna Austria), 2.5 mM Glutamax (Gibco/Life Technologies, Thermo Fisher Scientific), 5 ng/mL epidermal growth factor (Corning Inc, Corning, USA), and 16 mM HEPES (Merck/Sigma Aldrich, Vienna, Austria). The medium was further supplemented with 10% heat-inactivated (30 min at 56 °C) fetal bovine serum FBS (Gibco/Life Technologies, Thermo Fisher Scientific) and 1% penicillin–streptomycin (Merck/Sigma Aldrich) directly before use. Cultures were incubated at 39 °C and 10% CO2 under a humidified atmosphere (CO2 incubator, Binder, Tuttlingen, Germany) and sub-cultivated upon exceeding 90% confluency in the cultivation vessels. Routine testing for mycoplasma contaminations was conducted by PCR (Venor® GEM Classic Mycoplasma Detection Kit, Minerva Biolabs, Berlin, Germany).
10
Culturing Colorectal Cancer Cell Lines
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All cell lines (Caco-2, HT29, SW480, DLD1, HCT116, LS174T, RKO) were cultured in a humidified atmosphere (37°C, 5% CO2) in their corresponding medium supplemented with 10% fetal calf serum (Life Technologies) and 1% Glutamin (GE Healthcare). Caco-2, LS174T, RKO and SW480 were cultured in DMEM, high glucose, DLD1 in RPMI, HCT116 and HT29 in McCoys 5A medium (all Life Technologies). All CRC cell lines were screened for mycoplasma contamination using Venor GeM Classic Mycoplasma Detection Kit (Minerva biolabs) and DNA fingerprinting was performed for all seven cell lines at the Leibniz Institute DSMZ, Braunschweig, Germany.
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