Mycoalert kit

Manufactured by Promega

The MycoAlert® Kit is a rapid biochemical test kit used to detect the presence of mycoplasma contamination in cell cultures. The kit utilizes a bioluminescent enzyme reaction to measure the activity of mycoplasma-specific enzymes, providing a sensitive and reliable method for the detection of mycoplasma in cell culture samples.

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7 protocols using mycoalert kit

Insulin Mimetic Potential of Zincoforms

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In the present study, the 3T3-L1 cell line (mouse pre-adipocytes) was employed to assess the insulin mimetic potential of the title complex zincoforms. The employed cell line is an established in vitro model for the study of chemically induced cell differentiation toward adipogenesis. Cells (both premature and mature adipocytes) were cultured in 75 cm² cell culture flasks, under appropriately chosen conditions (5% CO₂ at 37 ^oC and standard humidity), in Dulbecco’s modified Eagle’s medium DMEM (Sigma, Steinheim, Germany). Culture media were supplemented with 10% Fetal Bovine Serum (FBS) (Biochrom, Berlin, Germany) and 1% penicillin–streptomycin (Biochrom, Berlin, Germany) prior to use. The specific cell line was tested and found free of mycoplasma contamination using the MycoAlert^® Kit (Promega). All experiments were run at least in triplicate, employing cells with a low passage number (P5–P9).

Gabriel C., Tsave O., Yavropoulou M.P., Architektonidis T., Raptopoulou C.P., Psycharis V, & Salifoglou A. (2021). Evaluation of Insulin-Like Activity of Novel Zinc Metal–Organics toward Adipogenesis Signaling. International Journal of Molecular Sciences, 22(13), 6757.

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Culturing and Characterization of HT-29 Colon Adenocarcinoma Cells

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The human colon adenocarcinoma cell line HT-29 was obtained from American Type Culture Collection (ATCC^® HTB-38™) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% L-Glutamine and 1% pen/strep antibiotic solution (growth medium) in humidified atmosphere at 37°C in 5% CO2. Cells were routinely tested for Mycoplasma through MycoAlert^® Kit (Promega). HT-29 derived from a primary tumor of a 44-year-old caucasian female, using the explant culture method. HT-29 show epithelial behavior in vitro forming a tight monolayer, while exhibiting similarity to enterocytes from the small intestine. They have tumorigenic potential and are positive for expression of c-myc, K-ras, H-ras, N-ras, Myb, sis and fos oncogenes. The p53 antigen is overproduced, and there is a G/A mutation in codon 273 of the p53 gene resulting in an Arg/His substitution.

Moracci L., Sensi F., Biccari A., Crotti S., Gaio E., Benetti F., Traldi P., Pucciarelli S, & Agostini M. (2022). An investigation on [5 fluorouracil and epigallocatechin-3-gallate] complex activity on HT-29 cell death and its stability in gastrointestinal fluid. Oncotarget, 13, 476-489.

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Breast Cancer Cell Line Characterization

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MCF7, ZR-75–1 and T47D cell lines were purchased from the ATCC (LGC Standards S.r.l., Milan, Italy). Cells were authenticated by DNA fingerprinting and isozyme detection. Cells were passaged for less than 6 months before their resuscitation for this study. All of our cell lines were routinely tested for mycoplasma contamination by Mycoalert Kit (Promega). Recombinant human IGF1 and recombinant human leptin were purchased from Peprotech. Insulin (Humulin R) was obtained from the Pharmacy of the IRCCS Ospedale Policlinico San Martino. Puromycin, protease/phosphatase inhibitor cocktail, β-oestradiol, sulforhodamine B, tamoxifen and fulvestrant (for in vitro use) were purchased from Sigma Aldrich S.r.l. fulvestrant for in vivo use was purchased from AstraZeneca (Faslodex). Palbociclib for in vitro experiments was purchased from Selleck Chemicals, while that for in vivo experiments was purchased from Medchem Express. 17β-oestradiol-releasing pellets were purchased from Innovative Research of America.

Caffa I., Spagnolo V., Vernieri C., Valdemarin F., Becherini P., Wei M., Brandhorst S., Zucal C., Driehuis E., Ferrando L., Piacente F., Tagliafico A., Cilli M., Mastracci L., Vellone V.G., Piazza S., Cremonini A.L., Gradaschi R., Mantero C., Passalacqua M., Ballestrero A., Zoppoli G., Cea M., Arrighi A., Odetti P., Monacelli F., Salvadori G., Cortellino S., Clevers H., De Braud F., Sukkar S.G., Provenzani A., Longo V.D, & Nencioni A. (2020). Fasting-mimicking diet and hormone therapy induce breast cancer regression. Nature, 583(7817), 620-624.

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Cell Lines and Treatments Protocol

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Human NB (SK‐N‐BE(2), SK‐N‐AS, SK‐N‐SH, SH‐EP, SH‐SY5Y) and mammary epithelial MCF 10A (non‐transformed) cells were acquired from the American Type Culture Collection. Human c‐Myc^−/− HEK293T cell line was acquired from EdiGene Biotechnology Inc. The cell lines were verified for authenticity by short tandem repeat loci, and were utilised for less than 6 months after being revived from frozen samples. Mycoplasma detection was routinely assessed with the MycoAlert Kit (Promega). The cells were cultivated in Dulbecco's modified Eagle's medium, which was supplemented with 10% foetal bovine serum from Gibco. Additionally, the cells were subjected to treatment with L‐proline, glutamate, leucine or lobeline (Sigma).

Wang J., Hong M., Cheng Y., Wang X., Li D., Chen G., Bao B., Song J., Du X., Yang C., Zheng L, & Tong Q. (2024). Targeting c‐Myc transactivation by LMNA inhibits tRNA processing essential for malate‐aspartate shuttle and tumour progression. Clinical and Translational Medicine, 14(5), e1680.

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ALCL Cell Lines Viability Assay

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ALCL cell lines Karpas299, SUDHL1, JB6, SR-786, SUP-M2, FE-PD, MAC1, and MAC2A were obtained from ATCC. All cell lines were cultured in RPMI-1640 supplemented with 10% FBS and 100 IU/mL penicillin. Cells were tested for mycoplasma every 3 months with the Mycoalert kit (Promega), and identity was confirmed by STR profiling (DFCI molecular diagnostics laboratory). Cell proliferation assays were performed by seeding 5000 cells per well in 96-well plates containing DMSO, crizotinib, alectinib, iberidomide, Stattic, or STAT3-IN-3 (MedChemExpress, LLC). Cell viability was assayed with CellTiter-Glo according to the manufacturer’s protocol (Promega).

Prutsch N., He S., Berezovskaya A., Durbin A.D., Dharia N.V., Maher K.A., Matthews J.D., Hare L., Turner S.D., Stegmaier K., Kenner L., Merkel O., Look A.T., Abraham B.J, & Zimmerman M.W. (2024). STAT3 couples activated tyrosine kinase signaling to the oncogenic core transcriptional regulatory circuitry of anaplastic large cell lymphoma. Cell Reports Medicine, 5(3), 101472.

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Quantification of miR-15a Expression in Human Myeloma Cell Lines

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Human myeloma cell lines have been previously described(49 (link)) and were maintained in RPMI-1640 supplemented with 5% FBS and glutamine, without antibiotics. None of these lines are listed on the ICLAC database of commonly misidentified cell lines and are routinely fingerprinted by assessment of copy number polymorphisms by PCR and tested for mycoplasma contamination using the MycoAlert® kit (Promega). The copy number for MIR15A in HMCL was extracted from the segmented copy number data previously described(49 (link)) using IGV, Gitools Heatmap, Export Gene Matrix TDM(50 (link)). RNA was extracted from five million logarithmically growing cells using the MirVana miRNA isolation kit (Invitrogen) following manufacturer’s instruction. Relative MIR15A expression was normalized to the average of U6 snRNA and RNU48 snoRNA (assay 01006) expression and quantified using the 2^-^dCt method. Each sample was tested in triplicates.

Chesi M., Stein C.K., Garbitt V.M., Sharik M.E., Asmann Y.W., Bergsagel M., Riggs D.L., Welsh S.J., Meermeier E.W., Kumar S.K., Braggio E, & Bergsagel P.L. (2020). Monosomic Loss of MIR15A/MIR16-1 Is a Driver of Multiple Myeloma Proliferation and Disease Progression. Blood Cancer Discovery, 1(1), 68-81.

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Rainbow Trout Liver Cell Culture

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2.1. Cells, culture media and growth conditions The RTL-W1 cell line derived from the liver of a 4 year old male rainbow trout (Lee et al., 1993) was kindly provided by S. Bony (INRA, France). Cells were routinely grown at 19 °C in 75 cm2 flasks (Easyflasks Nunc, Life Technology, USA) containing 15 mL of Leibovitz's L15 medium supplemented with 5% foetal bovine serum (FBS: PAA Laboratories, France) and 1% antibiotics (100 IU/mL penicillin, 100 μg/mL streptomycin). The culture medium was renewed every two days. When 80% confluence was reached, i.e. about once a week, cells were detached with trypsin 0.125% in phosphate-buffered saline supplemented with 0.03% ethylenediamine tetraacetic acid (PBS-EDTA) before being subcultured into a new 75 cm2 flask. Cell counts and routine viability measurements were made on a Bürker cell in presence of Trypan Blue as an exclusion dye. Cells were regularly checked for the absence of mycoplasma (MycoAlert kit, Promega, The Netherlands).

Ferain A., Bonnineau C., Neefs I., Rees J.F., Larondelle Y., Schamphelaere K.A, & Debier C. (2016). The fatty acid profile of rainbow trout liver cells modulates their tolerance to methylmercury and cadmium. Aquatic toxicology (Amsterdam, Netherlands), 177.

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