Human breast epithelial MCF-10A cells, mouse macrophage Raw 264.7 cells, and human adult keratinocyte HaCaT cells were obtained from the American Type Culture Collection. All cells have been passaged directly from the original low-passage stocks and were used before passage 30. The cells were also tested within the last three months for correct morphology under microscope and tested to detect any mycoplasma contamination using an e-Myco™ plus mycoplasma PCR detection kit (iNtRON Biotechnology, Seongnam, Korea). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
E myco plus mycoplasma pcr detection kit
Manufactured by iNtRON Biotechnology
Sourced in United States
The E-Myco™ plus Mycoplasma PCR Detection Kit is a laboratory equipment product developed by iNtRON Biotechnology. It is designed to detect the presence of mycoplasma contamination in cell cultures through a polymerase chain reaction (PCR) technique.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Bxpc 3, by American Type Culture Collection (2 mentions)
Aspc 1, by American Type Culture Collection (2 mentions)
Suit 2, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (2 mentions)
Rpmi 1640, by Nacalai Tesque (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Raw264.7 cell, by American Type Culture Collection (1 mentions)
Hacat cells, by American Type Culture Collection (1 mentions)
Mcf10a cells, by American Type Culture Collection (1 mentions)
G spin total dna extraction kit, by iNtRON Biotechnology (1 mentions)
Lymphocyte separation medium, by Lonza (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
Antibiotic antimycotic anti anti, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Mia paca2, by Cell Resource Center (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Sw1990, by American Type Culture Collection (1 mentions)
18 protocols using e myco plus mycoplasma pcr detection kit
1
Cell Line Characterization and Maintenance
2
Genomic DNA Extraction and Mycoplasma Test
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Genomic DNA was extracted using the G-Spin Total DNA Extraction Kit (iNtRON Biotechnology Inc.), and the mycoplasma test was performed using the e-Myco™ plus Mycoplasma PCR Detection Kit (iNtRON Biotechnology, Inc.) according to the manufacturer's protocol. Sample control and internal control bands indicate that sample preparation and PCR amplification were appropriately conducted, respectively.
3
Generation of Dendritic Cells from PBMCs
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PBMCs derived from apheresis were further enriched by density gradient centrifugation in lymphocyte separation medium (Lonza, Basel, Switzerland). The PBMCs were incubated for 2 h at 37 °C in X-VIVO15 medium (Lonza) in a plastic flask, and adherent cells were cultured in X-VIVO15 medium containing 2 % heat-inactivated autologous plasma, 1000 U/mL human interleukin-4 (IL-4, GMP-grade, Strathmann Biotec AG, Hannover, Germany), and 500 U/mL granulocyte macrophage colony-stimulating factor (GM-CSF, GMP-grade, GENTAUR Belgium BVBA, Kampenhout, Belgium). On day 6, loosely attached or floating immature DCs were collected. The immature DCs were stored in the gas phase of a liquid nitrogen tank until use. No bacteria, fungus, mycoplasma, or endotoxin contamination were detected in any cell culture products. The Gram’s iodine stain method was used for bacteria contamination evaluation. The detection of bacteria and fungus contamination was further performed by a growth-based rapid microbiological method with the BacT/ALERT automatic culture system (bioMerieux SA, Marcy I’Etoile, France). The detection of mycoplasma contamination was performed using a PCR-based method (e-Myco plus mycoplasma PCR detection kit, iNtRON Biotechnology, Kyungki-Do, Korea). The endotoxin contamination was determined using a Limius Amebocyte Lysate QCL-1000 Endotoxin test (Lonza).
4
NSCLC Cell Line Acquisition and Maintenance
5
Antibody Characterization in Pancreatic Cancer Cell Lines
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Antibodies against SLP-2 were purchased from ProteinTech Group, Inc. (cat. no. 10348-1-AP); GAPDH from Cell Signaling Technology, Inc. (product no. 2118S); GFPT2 from Abcam (product code ab190966); and anti-rabbit IgG as a secondary antibody (cat. no. A0545) was obtained from Sigma-Aldrich; Merck KGaA. The cell lines AsPC-1, BxPC-3, SUIT-2, and SW1990 were purchased from the American Type Culture Collection. The cell line PANC-1 was obtained from RIKEN and MIA-PaCa2 was provided from Cell Resource Center for Biomedical Research, Institute of Development, Aging and Center, Tohoku University (Sendai, Japan). Cells were expanded within 3 passages after being purchased, and multiple lots were stocked at −80°C. Mycoplasma contamination check tests were performed using e-Myco plus Mycoplasma PCR Detection Kit (iNtRON Biotechnology, Inc.). Cells were used at least <20 passages, but were not independently authenticated.
6
BHK-21 cell culture and GDVII virus infection
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BSR (single cell clone of BHK-21 cells, species Mesocricetus auratus, provided by Polly Roy, LSHTM, UK) were maintained in Dulbecco's modified Eagle's medium (DMEM), high glucose, supplemented with l -glutamine (1 mM), antibiotics, and fetal bovine serum (FBS) (5%), at 37°C and 5% CO2. Cells were verified as mycoplasma-free by PCR (e-Myco plus Mycoplasma PCR Detection Kit, iNtRON Biotechnology). Cells were seeded to achieve 80% confluence on the day they were infected with virus stocks listed in (23 (link)), based on GDVII isolate NC_001366 with three nucleotide differences present in WT and mutant viruses (G2241A, A2390G and G4437A; nt coordinates with respect to NC_001366). All infections, except for plaque assays, were carried out at a MOI of 3 in serum-free media, or media only for the mock-infected samples. After incubation at 37°C for 1 h, inoculum was replaced with serum-free media supplemented with FBS (2%), and infected cells were incubated at 37°C until harvesting or further processing.
7
Culturing A549 and A427 Lung Cancer Cells
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A549 (KCLB No. 10185) and A427 (KCLB No. 30053) cells were obtained from Korean Cell Line Bank (KCLB). Cells were cultured in DMEM (WelGENE Inc.) with 10% FBS (WelGENE Inc.) and antibiotics (100 units/mL of penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL of Fungizone; Life Technologies Corp.) at 37°C in a humidified atmosphere containing 5% CO2. Cells were periodically tested for Mycoplasma contamination (e-Myco plus Mycoplasma PCR Detection Kit, iNtRON Biotechnology).
8
Cultivation and Characterization of Cell Lines
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E.G‐7 (lymphoma) and B16‐F0 (melanoma) cell lines were purchased from the ATTC (Manassas, Virginia, USA). B16F10 was provided by the Cell Resource Center for Biomedical Research, Cell Bank, Institute of Development, Aging and Cancer, Tohoku University. Vero/N4 cells, which are NECTIN4‐expressing Vero cells, were established in this laboratory. E.G‐7 was maintained in complete RPMI 1640 medium (Sigma‐Aldrich) supplemented with 10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 55 μM 2‐mercaptethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin (cRPMI). B16‐F0, Plat‐A (kindly provided by Prof. T. Kitamura at Tokyo University, Tokyo, Japan), and Vero/N4 cells were cultured in DMEM (Sigma‐Aldrich) containing 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were cultured at 37°C with 5% CO2. All cell lines were tested for Mycoplasma sp. using the e‐Myco™ plus Mycoplasma PCR Detection Kit (iNtRON Biotechnology).
9
Culturing Pancreatic Cancer Cell Lines
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Cell lines were purchased from the American Type Culture Collection ATCC (MIAPaCa-2, PANC-1, Suit-2, BxPC-3 and AsPC-1). Pancreatic cancer cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM GlutaMAX) supplemented with 10% FBS. Primary CAFs were recovered from liquid nitrogen simultaneously and placed in a T75 flask with IMDM supplemented with 10% FBS, 2% L-glutamine and 1% penicillin–streptomycin. Both primary and established cells were incubated at 37 °C in a 5% CO2 air humidified atmosphere. Cells were validated by STR profiling and tested for mycoplasma using e-Myco plus mycoplasma PCR detection kit (iNTRON Biotechnology) following the manufacturer’s instructions.
10
Culturing Melanoma Cell Lines
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WM266-4 human and mouse 21015 melanoma cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% penicillin-streptomycin (ThemoFicher) and 10% FBS (ThermoFischer). The cultured cells were maintained at 5% CO2 in humidified chamber at 37°C.
Both cell lines were confirmed to be mycoplasma negative (e-Myco plus Mycoplasma PCR detection kit, iNTRON Biotechnology). Passaging was carried out using 0.25% Trypsin-EDTA (ThermoFischer) and resuspension in complete medium. Cell counting was performed using Countess automated cell counter with trypan blue exclusion (ThermoFischer).
Both cell lines were confirmed to be mycoplasma negative (e-Myco plus Mycoplasma PCR detection kit, iNTRON Biotechnology). Passaging was carried out using 0.25% Trypsin-EDTA (ThermoFischer) and resuspension in complete medium. Cell counting was performed using Countess automated cell counter with trypan blue exclusion (ThermoFischer).
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