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Pcr mycoplasma test kit 1 c

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The PCR Mycoplasma Test Kit I/C is a laboratory equipment designed for the detection of Mycoplasma contamination in cell cultures. It utilizes a polymerase chain reaction (PCR) method to amplify specific DNA sequences for the identification of Mycoplasma species.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions) L glutamine, by Thermo Fisher Scientific (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Mda mb 231, by American Type Culture Collection (3 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Merck Group (2 mentions) Penicillin, by Merck Group (2 mentions) Short tandem repeat genotyping, by Eurofins (2 mentions) Uplanfln, by Olympus (2 mentions) Color mosaik 18.2 camera, by Visitron (2 mentions) Lcachn, by Olympus (2 mentions) Gm12878, by Coriell Institute for Medical Research (2 mentions) Mcf 7, by American Type Culture Collection (2 mentions) Transferrin, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Harvard Bioscience (2 mentions) Mg 63, by American Type Culture Collection (2 mentions) Minimum essential medium (mem), by Biowest (2 mentions) Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin p s, by Thermo Fisher Scientific (2 mentions)

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PCR Mycoplasma Test (3 citations) Mycoplasma test kit (2 citations) PCR Mycoplasma Kit (2 citations)

33 protocols using pcr mycoplasma test kit 1 c

1

Characterization of C2C12 and HEK293T Cells

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C2C12 and HEK293T cells were obtained from the American Type Culture Collection (ATTC, Masnassas, Virginia) and analyzed for potential myoplasma contamination every two month using the PCR Mycoplasma Test Kit I/C from Promocell (Heidelberg, Germany). Identity of C2C12 cells was authenticated by induction of myotube differentiation. Antibodies recognizing BRAF, EEA1, LAMIN A/C and MYOGENIN were obtained from BD Biosciences (San Jose, California) while PAX3, MYOD and myosin heavy chain antibodies (MHC) were from the Developmental Studies Hybridoma bank (Iowa City, Iowa). Additional PAX3 and anti-LBX1 antibodies were purchased from Abcam (Cambridge, Massachusetts). Anti-phospho-PAX3 (Serine 205) was a kind gift of Dr. A. D. Hollenbach (Miller et al., 2008 (link)). The antibody against CD31 and recombinant Scatter Factor/Hepatocyte Growth factor (HGF) was obtained from R and D Systems (Minneapolis, Minnesota). Anti-phospho BRAF, anti-phospho ERK1/2 and anti-GAPDH antibodies were purchased from Cell Signaling (Danvers, Massachusetts). Anti-MYF5 was from Santa Cruz (Dallas, Texas) and Anti-GST was from GE healthcare (Chicago, Illinois).
Shin J., Watanabe S., Hoelper S., Krüger M., Kostin S., Pöling J., Kubin T, & Braun T. (2016). BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development. eLife, 5, e18351.
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2

Muller Cell and Pericyte Culture for Hyperglycemia Studies

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The human Müller cell line MIO-M1 was obtained from the UCL Institute of Ophthalmology (London, United Kingdom; ref. 51 (link)). PMCs from mitoQC+/+ mice were isolated and cultured as previously described (25 (link)). PMCs were used for experiments from P2–P6, where most cells showed a senescence phenotype. Cultures were maintained in DMEM (containing 10% FCS [Thermo Fisher Scientific], 100 U/mL penicillin-streptomycin [Sigma-Aldrich]) and supplemented with 5.5 mM D-glucose (NG), 30.5 mM D-glucose (Sigma-Aldrich; HG), or 30.5 mM LG (Alfa aesar; 25 mM LG + 5.5 mM NG) for 5 days. The selection of 30.5 mM D-glucose was based on the levels of hyperglycemia found in the plasma of Ins2Akita/+ mice. For mitophagy-induced amino acid starvation, cultures were maintained in HBSS (Thermo Fisher Scientific; 16 hours). Autophagy flux was blocked with 100 μM chloroquine (12 hours). Endpoint experiments were performed in 70%–80% confluent cultures. No mycoplasma was detected in the cell cultures (PCR Mycoplasma Test Kit I/C; PromoCell).
Hombrebueno J.R., Cairns L., Dutton L.R., Lyons T.J., Brazil D.P., Moynagh P., Curtis T.M, & Xu H. (2019). Uncoupled turnover disrupts mitochondrial quality control in diabetic retinopathy. JCI Insight, 4(23), e129760.
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3

Murine Embryonic Stem Cell Inhibitor Assay

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B6 mESCs used in this study were established and maintained in 2i/LIF medium as described in Pryzhkova and Jordan, 2016 (link); Pryzhkova et al., 2020 (link). mESCs were verified to be negative for mycoplasma using the PCR Mycoplasma Test Kit I/C (PromoCell). For cell growth analysis mESCs were cultured in the presence of 10 µM CHEK2 inhibitor II (Cayman), 3 µM CHEK1 inhibitor LY2603618 (Cayman), or p53 inhibitor cyclic pifithrin-alpha hydrobromide (Cayman), with or without 100 µM IAA for 48 hr. Drugs were added 18–20 hr after passaging. mESCs were counted at the time of passaging and after 48 hr of growth in the presence of drugs.
Atkins A., Xu M.J., Li M., Rogers N.P., Pryzhkova M.V, & Jordan P.W. (2020). SMC5/6 is required for replication fork stability and faithful chromosome segregation during neurogenesis. eLife, 9, e61171.
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4

Isolation and Culture of Cardiac Perivascular Cells

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Cardiac PCs were immunosorted as CD31neg/CD34pos cells from human myocardial samples, and expanded in a dedicated medium supplemented with human recombinant growth factors and 2% v/v foetal calf serum (FCS) (ECGM2 complete kit, C-22111, PromoCell) as previously described [11 (link),28 (link)]. Briefly, samples were finely minced using scissors and scalpel until nearly homogenous and digested with Liberase (Roche) for up to 1 h at 37 C, with gentle rotation. The digest was passed through 70-, 40-, and 30-μm strainers. Finally, the cells were recovered and sorted using anti-CD31 and -CD34 microbeads (Miltenyi) to deplete the population of CD31pos ECs and select CD31neg/CD34pos cells, which distinguish a population of perivascular cells in situ [11 (link),28 (link)]. After expansion to passage 3, the purity of the cell population was verified using immunocytochemistry (ICC) or flow cytometry [11 (link),28 (link)].
Human coronary artery ECs (CAECs) were purchased from PromoCell and expanded in the same medium used for PCs. All cells used in the present study tested negative for mycoplasma contamination (assessed using the PCR Mycoplasma Test Kit I/C, PromoCell, cat# PK-CA91-1096). Cells were used between passages 4 and 7.
Avolio E., Carrabba M., Milligan R., Kavanagh Williamson M., Beltrami A.P., Gupta K., Elvers K.T., Gamez M., Foster R.R., Gillespie K., Hamilton F., Arnold D., Berger I., Davidson A.D., Hill D., Caputo M, & Madeddu P. (2021). The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease. Clinical Science (London, England : 1979), 135(24), 2667-2689.
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5

Characterization of B16F1 GFP Melanoma Cells

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B16F1 GFP cells [18 (link)] carry deletions of exons 1α, 1β, and 2 of the Ink4a/Arf gene locus, resulting in loss of p16Ink4a and p19Arf protein expression and altered p53 protein expression without detectable gene mutations, however with no activating BRAF mutations in exons 11 and 15 [19 (link)]. B16F1 cells were grown in DMEM medium (+4.5 g/L D-Glucose, L-glutamine, Thermo Fisher Scientific, Darmstadt, Germany) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were counted using a Neubauer cell counting chamber (Laboroptik, Friedrichsdorf, Germany). Cells were routinely tested negative for mycoplasma (PCR Mycoplasma Test Kit I/C; PK-CA91-1096; Promocell, Heidelberg, Germany). For coating, the recombinant proteins (acid extracted Bovine Collagen Type I, Curacyte Discovery GmbH, Leipzig, Germany; human fibronectin, Roche Life Science, Penzberg, Germany) were diluted in PBS and incubated overnight at 4 °C.
Pach E., Brinckmann J., Rübsam M., Kümper M., Mauch C, & Zigrino P. (2021). Fibroblast MMP14-Dependent Collagen Processing Is Necessary for Melanoma Growth. Cancers, 13(8), 1984.
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6

Characterizing E/R+ REH Cell Line

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The E/R+ REH cell line (ACC-22, DSMZ, Germany) was maintained in RPMI 1640 (Gibco, Thermo Fisher) supplemented with 10% FBS (Gibco, Thermo Fisher), 2 mM L-glutamine (Gibco, Thermo Fisher), penicillin (100 U/ml), and streptomycin (100 mg/ml) (Sigma-Aldrich).
Mycoplasma status was defined negative for all cell lines by PCR (PCR Mycoplasma Test Kit I/C, PromoCell GmbH, Germany) and cell lines were authenticated by Short Tandem Repeat genotyping (Eurofins Genomics, Ebersberg, Germany).
Mehtonen J., Teppo S., Lahnalampi M., Kokko A., Kaukonen R., Oksa L., Bouvy‐Liivrand M., Malyukova A., Laukkanen S., Mäkinen P.I., Rounioja S., Ruusuvuori P., Sangfelt O., Lund R., Lönnberg T., Lohi O., & Heinäniemi M. (2020). Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1 positive pediatric leukemia identifies drug-targetable transcription factor activities.
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7

Mycoplasma Detection by PCR Imaging

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Mycoplasma PCR was performed using the PCR Mycoplasma Test Kit I/C (PK-CA91-1024, PromoCell, Heidelberg, Germany) according to the manufacturer's instructions. The kit includes a Olympus) equipped with a Color Mosaik 18.2 camera (Visitron Systems) and the SPOT Advanced software (version 4.6.3.8) . Every time, overview (40x magnification: Olympus UPlanFLN, 4x/0.3 PhP) and close-up pictures (200x magnification: Olympus LCAchN, 20x/0.40 PhP) were taken and archived.
Tigges J. (2021). Academic application of Good Cell Culture Practice for induced pluripotent stem cells_suppl. ALTEX.
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8

Culturing E/R+ REH Cell Line

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The E/R+ REH cell line (ACC-22, DSMZ, Germany) was maintained in RPMI 1640 (Gibco, Thermo Fisher) supplemented with 10% FBS (Gibco, Thermo Fisher), 2 mM l-glutamine (Gibco, Thermo Fisher), penicillin (100 U/ml), and streptomycin (100 mg/ml) (Sigma-Aldrich). Mycoplasma status was defined negative for all cell lines by PCR (PCR Mycoplasma Test Kit I/C, PromoCell GmbH, Germany), and cell lines were authenticated by Short Tandem Repeat genotyping (Eurofins Genomics, Ebersberg, Germany).
Mehtonen J., Teppo S., Lahnalampi M., Kokko A., Kaukonen R., Oksa L., Bouvy-Liivrand M., Malyukova A., Mäkinen A., Laukkanen S., Mäkinen P.I., Rounioja S., Ruusuvuori P., Sangfelt O., Lund R., Lönnberg T., Lohi O, & Heinäniemi M. (2020). Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities. Genome Medicine, 12, 99.
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9

Culturing BT-474 Breast Cancer Cells

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BT-474 cells (ATCC number HTB-20) were cultured in complete MEM (16140071, Gibco, LifeTechnologies) supplemented with 10% fetal bovine serum (FBS) (P40-37500, PAN-Biotech) and 1% penicillin/streptomycin (10000 U mL−1, 15140122, ThermoFisher Scientific). Culture Medium was changed every 3–4 days, passaged at ∼80% confluency using 0.05% Trypsin-EDTA (15400-045, Gibco, LifeTechnologies). Absence of mycoplasma contamination from BT-474 during the experiments was confirmed by using the PCR mycoplasma Test Kit I/C (PromoCell).
Rodriguez-Perdigon M., Haeni L., Rothen-Rutishauser B, & Rüegg C. (2023). Dual CSF1R inhibition and CD40 activation demonstrates anti-tumor activity in a 3D macrophage- HER2+ breast cancer spheroid model. Frontiers in Bioengineering and Biotechnology, 11, 1159819.
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10

Melanoma and Leukemia Cell Culture Protocol

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Target cells including K562 and melanoma cell lines were kept in culture for 2–3 months (24–30 passages). The leukemia cell line K562 (ATCC, #CCL-243) was cultured in RPMI1640 medium (Thermo Fisher Scientific, #21875–034) supplemented with 10% FCS. Melanoma cell lines were maintained in melanoma growth medium [TU2%, consisting of 80% MCDB153-Basalmedium (Biochrom AG, #F 8105), 20% L-15, Leibowitz-Medium (PromoCell, #C-24300), 2% FCS, 1.68 mmol/L CaCl2 (Sigma-Aldrich, #21115), 5 ng/mL human insulin (Sigma-Aldrich, #I9278)]. The 1205Lu cell line was cultured in TU2% with 200 mmol/L L-glutamine and without insulin. The 15 melanoma cell lines used in this study have been provided, authenticated, and documented by the Herlyn laboratory (Wistar Institute, Philadelphia, PA; see Supplementary Table S1 and Resources at https://wistar.org/our-scientists/meenhard-herlyn). All cell lines were regularly tested for Mycoplasma contamination by using PCR Mycoplasma Test Kit I/C (PromoCell #PK-CA91–1024) according to the manufacturer’s manual. Protein expression in melanoma cells was manipulated by using siRNA or DNA transfection or by CRISPR-Cas9. Immunoblotting, IHC, immunofluorescence, and flow cytometry were applied to determine protein abundance. For full description of the methods used, please see Supplementary Data.
Cappello S., Sung H.M., Ickes C., Gibhardt C.S., Vultur A., Bhat H., Hu Z., Brafford P., Denger A., Stejerean-Todoran I., Köhn R.M., Lorenz V., Künzel N., Salinas G., Stanisz H., Legler T., Rehling P., Schön M.P., Lang K.S., Helms V., Herlyn M., Hoth M., Kummerow C, & Bogeski I. (2021). Protein Signatures of NK Cell–Mediated Melanoma Killing Predict Response to Immunotherapies. Cancer research, 81(21), 5540-5554.
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