Mycoplasma detection kit

Manufactured by Applied Biological Materials

Sourced in Canada

The Mycoplasma detection kit is a laboratory equipment product designed to detect the presence of mycoplasma contamination in cell cultures. The kit provides a reliable method for the identification of mycoplasma species through a series of tests and procedures.

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10 protocols using mycoplasma detection kit

Characterization of Ireb2 Knockout MEFs

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Ireb2 KO MEFs and wild type MEFs isolated from littermate control were generous gifts from Dr. Tracey Rouault (NICHD, NIH). Mouse embryonic fibroblasts were cultured in Dulbecco's Modified Eagle's Medium (D MEM, Corning) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin. We have verified that Ireb2 was knockout in the Ireb2 KO MEFs. HEK293 cells were purchased from ATCC and cultured in Minimum Essential Medium (MEM, Corning) supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 1 mM sodium pyruvate. We have not performed separate verification since receiving the HEK293 cell line from ATCC. The cell lines were regularly using PCR-based mycoplasma detection kit (ABM) and were negative for mycoplasma contamination.

Chang H.C., Shapiro J.S., Jiang X., Senyei G., Sato T., Geier J., Sawicki K.T, & Ardehali H. (2021). Augmenter of liver regeneration regulates cellular iron homeostasis by modulating mitochondrial transport of ATP-binding cassette B8. eLife, 10, e65158.

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Cell Culture Protocols for Zebrafish, HaCaT, and HDF

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The zebrafish PAC2^{57 (link)} cell line was propagated at 26 °C in an atmospheric CO₂, non-humidified cell culture incubator; cells were cultured in L-15 (Leibovitz) medium (Gibco BRL) supplemented with 15% Fetal Bovine Serum (Sigma-Aldrich, St Louis, MO), 100 units/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml gentamicin (Gibco BRL).
HaCaT (human spontaneously immortalized keratinocytes from adult skin) and HDF (human primary dermal fibroblast) were purchased from Cell Line Service (CLS, Germany) and cultured in a humidified incubator at 37 °C and 5% CO₂ in DMEM High Glucose (Gibco BRL) supplemented with 10% Fetal Bovine Serum (Gibco BRL), 1% L-Glutamine (Gibco BRL) and 1% Pen-Strep solution (Gibco BRL).
Cells were routinely checked for mycoplasma contamination, using a mycoplasma detection kit (abm, Canada).

Guarino A.M., Mauro G.D., Ruggiero G., Geyer N., Delicato A., Foulkes N.S., Vallone D, & Calabrò V. (2019). YB-1 recruitment to stress granules in zebrafish cells reveals a differential adaptive response to stress. Scientific Reports, 9, 9059.

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Culturing and Expanding Stem Cells

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HEK293T cells (American Type Culture Collection, CRL-3216) were cultured in the T-75 flask (Corning) using high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX and sodium pyruvate (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1× penicillin-streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO₂. Upon reaching 80 to 90% confluency, cells were dissociated using TrypLE Express (Life Technologies) and passaged at a ratio of 1:3. Cells were verified mycoplasma-free using a mycoplasma detection kit (abm). ESI-017 hESCs (ESI BIO, CVCL_B854) and iPSCs (Coriell Institute, AICS-0058-067) were maintained in mTeSR1 (STEMCELL Technologies) in tissue culture dish coated with Matrigel (1:200; Corning). Dispase (STEMCELL Technologies) was used for routine passage. To perform nucleofection, a single-cell suspension was prepared using Accutase (Innovative Cell Technologies). The pluripotency of those cells was confirmed via staining of Oct4, Sox2, and Nanog. Both ESI-017 and iPSC lines were routinely tested for mycoplasma contamination and found negative.

Lee S., Ding N., Sun Y., Yuan T., Li J., Yuan Q., Liu L., Yang J., Wang Q., Kolomeisky A.B., Hilton I.B., Zuo E, & Gao X. (2020). Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects. Science Advances, 6(29), eaba1773.

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Hypoxia Treatment of NSCLC Cell Lines

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The human NSCLC cell lines A549 and H1650 were purchased from ATCC. A549 cells were maintained in Ham’s F12K medium (Cell Gro, Corning) supplemented with 10% fetal bovine serum (VWR Life Science Seradigm, Radnor, PA) while H1650 cells were grown in RPMI 1640 (Gibco, Life Technologies) containing 10% FBS. All the cultures were maintained at 5% CO₂ at 37^oC. For the hypoxia treatment, the cells were subjected to 1% O₂ with 5% CO₂ at 37^oC for 24 hours. All the cells were routinely tested for mycoplasma contamination using a PCR-based Mycoplasma Detection Kit (abm, #G238; Richmond, BC, Canada). Cell lines were authenticated using human STR sequencing analyses conducted by the Molecular Genomics Core at Moffitt Cancer Center; cultured cells retrieved from the frozen stocks were discarded after every 10–15 passages.

Bora-Singhal N., Saha B., Mohankumar D., Padmanabhan J., Coppola D, & Chellappan S. (2022). A Novel PHD2/VHL-mediated Regulation of YAP1 Contributes to VEGF Expression and Angiogenesis. Cancer Research Communications, 2(7), 624-638.

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Fibroblast Culture and Characterization

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Fibroblasts were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Waltham). Fibroblasts were used between passages 8 and 11 (Supplemental Table S1). Experiments were always run with affected cells and either contralateral unaffected cells or healthy control cells in parallel, as applicable.
Variant allele frequency (VAF) was determined by ddPCR as previously described (Kang H 2018 (link)). In these experiments, VAF for affected cells was between 11 and 42% for affected fibroblasts, and for unaffected and healthy control cells was always 0% (Supplemental Table S1).
Human dermal microvascular endothelial cells (HDMVECs) were purchased from American Type Cell Culture (ATCC, Manassas) or PromoCell and grown in endothelial cell growth medium-2 with all BulletKit supplements (EGM-2, Lonza, Switzerland). HDMVECs were used for experiments at passages 3–4.
Cells were routinely tested for mycoplasma infection using a mycoplasma detection kit according to the manufacturer’s protocol (abm, Canada). All experiments are shown for cells which were mycoplasma-free. Note that Melo 6 cells (affected and unaffected) initially had a mycoplasma infection, which was treated with Plasmocin (Invivogen, San Diego) and found to be negative during all future testing.

Hurley-Novatny A.C., Allbritton-King J.D., Jha S., Cowen E.W., Colbert R.A., Navid F, & Bhattacharyya T. (2022). Fibroblasts from Melorheostosis Patients Promote Angiogenesis in Healthy Endothelial Cells through Secreted Factors. The Journal of investigative dermatology, 142(9), 2406-2414.e5.

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Characterization of Cell Lines for Cancer Research

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HaCaT, TU212, and TU686 cells were purchased from iCell Biotechnology Co., LTD. (Shanghai, China). All three cells had STR testing certificates (Fig. S1). According to Mycoplasma Detection Kit (#G238; abm, Canada), all cell lines were negative for mycoplasma (Fig. S2). TU212 is a laryngeal squamous cell carcinoma cell line and TU686 is a hypopharyngeal carcinoma cell line. TU212 and TU686 cells were cultured in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere containing 5% CO₂. Prior to subsequent experiments, cells were treated with MK2206 (5um), a highly selective Akt inhibitor, for 12 h.

Zhu Q., Zhang X., Lu F., Miao S., Zhang C., Liu Z., Gao Z., Qi M., An X., Geng P., Wang S., Ren H., Han F., Zhang R, & Zha D. (2024). RUNX1-BMP2 promotes vasculogenic mimicry in laryngeal squamous cell carcinoma via activation of the PI3K-AKT signaling pathway. Cell Communication and Signaling : CCS, 22, 227.

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Cytotoxicity Evaluation of Natural Compounds

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HaCaT (human spontaneously immortalized keratinocytes from adult skin) were purchased from Cell Line Service (CLS, Hattersheim am Main, Germany). Cells were cultured in DMEM High Glucose (Gibco BRL Thermo Fisher, Milan, Italy) supplemented with 10% Fetal Bovine Serum (Gibco BRL, Thermo Fisher, Milan, Italy), 1% L-Glutamine (Gibco BRL) and 1% Pen-Strep solution (Gibco BRL) in a humidified incubator at 37 °C and 5% CO₂. Cells were routinely checked for mycoplasma contamination, using a mycoplasma detection kit (ABM, Vancouver, Canada). Cytotoxicity was determined by the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (Sigma-Aldrich, St Louis, MO, USA). HaCaT were seeded in 24-well plates at 3.0 × 10⁴ per well and treated with sphaeropsidin A, sphaeropsidone, and epi-epoformin at concentrations between 3.125 µg/mL and 100 µg/mL for 24 h. The assay was performed according to the manufacturer’s instructions. The optical absorbance was determined at 570 nm and 630 nm using an iMark microplate reader (Bio-Rad, Milan, Italy). Each value shown in the plot is mean ± SD of triplicate determinations. Asterisks represent significant results (*** p > 0.001; **** p < 0.0001).

Roscetto E., Masi M., Esposito M., Di Lecce R., Delicato A., Maddau L., Calabrò V., Evidente A, & Catania M.R. (2020). Anti-Biofilm Activity of the Fungal Phytotoxin Sphaeropsidin A against Clinical Isolates of Antibiotic-Resistant Bacteria. Toxins, 12(7), 444.

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Culturing Hepa 1-6 Cells for Research

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Hepa 1–6 cells, originally sourced from the ATCC (RRID: CVCL_0327), were maintained in DMEM (319-005-CL, Wisent) supplemented with 10% (v/v) fetal bovine serum (FBS, 12483020, Thermo Fisher Scientific) and 100 units/ml penicillin-streptomycin (450-201-EL, Wisent). Cultured cells were maintained at 37 °C with 5% CO₂, and mycoplasma contamination was regularly assessed using the polymerase chain reaction (PCR) mycoplasma detection kit (G238, ABM).

Scholtes C., Dufour C.R., Pleynet E., Kamyabiazar S., Hutton P., Baby R., Guluzian C, & Giguère V. (2024). Identification of a chromatin-bound ERRα interactome network in mouse liver. Molecular Metabolism, 83, 101925.

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Establishing Syngeneic Mouse Breast Cancer Model

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The mouse breast cancer cell line E0771 was obtained from ATCC (Manassas, VA, USA) and cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS. Mycoplasma infection was excluded by a PCR test of the culture media using a mycoplasma detection kit (G238, Applied Biological Materials Inc, Richmond, BC, Canada). IOA-289 was supplied by iOnctura (Genève, Switzerland). Rabbit anti-CD45 (70257), rabbit anti-F4/80 (70076), rabbit anti-CD8α (98941), rabbit anti-CD206 (24595), rabbit anti-FoxP3 (12653), and rabbit anti-αSMA (19245) antibodies were from Cell Signaling Technology (Danvers, MA, USA), and Rabbit anti-CD31 antibody (ab182981) was from Abcam Inc. (Toronto, ON, Canada).

Tang X., Morris A.J., Deken M.A, & Brindley D.N. (2023). Autotaxin Inhibition with IOA-289 Decreases Breast Tumor Growth in Mice Whereas Knockout of Autotaxin in Adipocytes Does Not. Cancers, 15(11), 2937.

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Cell Culture Protocols for HEK293T and U87 Cells

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HEK293T (Thermo Fisher Scientific) and U87 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco, 41965039) supplemented with 10% foetal bovine serum (FBS; Gibco, 10270106), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco, 5070063). 4EHP-KO and GIGYF2-KO HEK293 cells have been described previously (Xu et al., 2022 (link); Zhang et al., 2021 (link)). Parental, 4EHP-KO and GIGYF2-KO HEK293 cells were maintained in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 100 µg/ml zeocin (Invitrogen, 460509) and 15 µg/ml blasticidin (BioShop, BLA477). All cells were cultured at 37°C in a humidified atmosphere with 5% CO₂ and regularly tested for the presence of mycoplasma contamination using a mycoplasma detection kit (Applied Biological Materials, G238).

Naeli P., Zhang X., Snell P.H., Chatterjee S., Kamran M., Ladak R.J., Orr N., Duchaine T., Sonenberg N, & Jafarnejad S.M. (2023). The SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression. Journal of Cell Science, 136(19), jcs261286.

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