HEK293 cells were cultured routinely in DMEM supplemented with 10% (v/v) fetal bovine serum in humidified 5% (v/v) CO2 in air at 37 °C. Cells were seeded into 35-mm FluoroDishTM dishes (World Precision Instruments) and transfected after 24 h using PEI. 2 μg of DNA was added to 100 μl of pre-warmed serum-free DMEM followed by 12 μl of 1 mg/ml PEI (pH 7.4). The mixture was incubated at room temperature for 10 min. 600 μl of pre-warmed growth media was added, and cell culture media were replaced with the transfection mix. 2 ml of growth media was added after 2 h. The transfection mix was replaced by 2 ml of culture medium after a further 22 h. Transfected cells were imaged 24–36 h after transfection. Rat primary cortical astrocytes were isolated and cultured as described previously (12 (link)). All cells tested negative for mycoplasma using the EZ-PCR mycoplasma test kit (Biological Industries).
Ez pcr mycoplasma test kit
Manufactured by Sartorius
Sourced in Israel, United States
The EZ-PCR Mycoplasma Test Kit is a laboratory equipment product designed to detect the presence of mycoplasma contamination in cell cultures. The kit utilizes a polymerase chain reaction (PCR) technique to amplify and identify specific mycoplasma DNA sequences. It provides a rapid and reliable method for mycoplasma detection in a laboratory setting.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
L glutamine, by Thermo Fisher Scientific (7 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Sartorius (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Sartorius (5 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by Merck Group (4 mentions)
Penicillin, by Sartorius (3 mentions)
Hct116, by American Type Culture Collection (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Lncap, by American Type Culture Collection (3 mentions)
Mda mb 231, by American Type Culture Collection (3 mentions)
Roswell park memorial institute (rpmi), by Sartorius (3 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Merck Group (2 mentions)
134 protocols using ez pcr mycoplasma test kit
1
Transfection and Imaging of HEK293 Cells
2
Transfection and RNA Polymerase II Inhibition Assay
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U2OS, HEK-293, and HEK-293T cell lines were obtained from ATCC and grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C.
For transfection, HEK-293 cells were plated on 35 mm plates in 40% confluency and grown for 16 h.
Transfection of pGL3 luciferase reporter vector was performed using TransIT-LT1 transfection reagent according to thr manufacturer’s instructions. Cells were collected for analysis 24 h after transfection.
Transfection of miRNA, synthetic miR-92a-3p, let-7, or negative control dsRNA was performed using TransIT-X2TM Dynamic Delivery System according to the manufacturer’s instructions. Cells were collected for analysis 40 h after transfection. Three replicates were used for this experiment.
To inhibit RNA polymerase II transcription activity, U2Os cells were incubated with α-amanitin (10 µg/ml; Sigma) for 6 or 9 h at 37 °C.
All cell lines were tested for mycoplasma contamination using EZ-PCR Mycoplasma Test Kit (Biological Industries; Cat. No. 20-700-20).
For transfection, HEK-293 cells were plated on 35 mm plates in 40% confluency and grown for 16 h.
Transfection of pGL3 luciferase reporter vector was performed using TransIT-LT1 transfection reagent according to thr manufacturer’s instructions. Cells were collected for analysis 24 h after transfection.
Transfection of miRNA, synthetic miR-92a-3p, let-7, or negative control dsRNA was performed using TransIT-X2TM Dynamic Delivery System according to the manufacturer’s instructions. Cells were collected for analysis 40 h after transfection. Three replicates were used for this experiment.
To inhibit RNA polymerase II transcription activity, U2Os cells were incubated with α-amanitin (10 µg/ml; Sigma) for 6 or 9 h at 37 °C.
All cell lines were tested for mycoplasma contamination using EZ-PCR Mycoplasma Test Kit (Biological Industries; Cat. No. 20-700-20).
3
Culturing Mouse Mammary Carcinoma Cells
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Met-1 mouse mammary gland carcinoma cells were a gift from J. Pollard (The University of Edinburgh, Scotland, UK). C18 primary mouse mammary gland carcinoma cells were prepared in our laboratory from fresh tumor tissue of MMTV-PyMT female mice (Sharon et al., 2015 (link)). The C166 endothelial cell line was purchased from the ATCC. All cell lines were routinely tested for mycoplasma using the EZ-PCR-Mycoplasma test kit (Biological Industries; 20-700-20).
4
Mycoplasma-Free Cell Line Characterization
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All cell lines were characterized and purchased from ATCC. Cells were used for experiment up to p20 and were Mycoplasma free, using EZ-PCR Mycoplasma Test Kit (Biological Industries, Catalog number: 2070020).
5
Melanoma Cell Injection Protocol
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Mouse melanoma cells B16F10 were grown as described [13 (link)] and extensively washed in phosphate buffered saline (PBS) prior to injection at 1.6 × 105 cells/100 μL. B16F10-luc cells (mouse melanoma cells expressing luciferase) were kindly provided by Dr. Victoria Brentville (Scancell, Nottingham, UK) and injected at a dose of 4 × 105 cells/100 μL. Cells were confirmed to be mycoplasma negative using the EZ-PCR Mycoplasma Test Kit (Biological Industries, Cromwell, CT, USA).
6
Cell Culture Protocols for Ovarian and Lung Cancer
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Human ovarian clear cell carcinoma ES2 cell line (American Type Culture Collection) was cultured in DMEM supplemented with 10% FBS. Mouse D122 Lewis lung carcinoma cell line (Eisenbach et al, 1984 (link)), kindly provided by Prof. Lea Eisenbach, (Weizmann Institute of Science, Israel), was grown in DMEM containing 10% FBS, 1 mM sodium pyruvate, and 1% nonessential amino acids. All cell lines were routinely tested for mycoplasma contamination using the EZ-PCR Mycoplasma Test Kit (Biological Industries).
7
Cell Line Expansion and Validation Protocol
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All cell lines were purchased from ATCC (Manassas, VA, USA). U87MG cells were purchased by Dr. Quezada-Monrás, while T98G and U251 cells were purchased and gently donated by Dr. Varas-Godoy. Once they arrived at the laboratory, the cells were immediately expanded in DMEM-F12 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Waltham, MA, USA) at 37 °C and 5% CO2, followed by storage in liquid nitrogen at −190 °C. Once a year, one N2 aliquot was thawed, expanded, and stored again at −80 °C. For experiments, one −80 °C aliquot was thawed and grown in normal media. All experiments were performed within one year, and cells were eliminated after 15 passages, as requested by each local biosecurity committee. Mycoplasma contamination was tested monthly using the EZ-PCR Mycoplasma Test kit (Biological Industries, Beit Haemek, Israel), with the last test being performed six months ago and yielding no contamination.
8
Mycoplasma Contamination Assessment
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The absence of mycoplasma was regularly assessed by EZ-PCR Mycoplasma Test Kit (Biological Industries, BI) following the manufacturer’s instruction.
9
Characterization of Vascular Endothelial Cells
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HUVECs (VECs) and HMVEC-dLyAd cells (LECs) were purchased from Lonza (Walkersville, MD, USA). All cells were characterized before use, mycoplasma-free, using an EZ-PCR Mycoplasma Test Kit (Biological Industries), and were used for the experiments up to passage 12. All cells were kept in a humidified incubator at 37 °C with 5% CO2. HUVECs were maintained in a specific medium supplemented with the PeproGrow-MacroV kit (ENDO-BM & GS-MacroV, PeproTech) and penicillin/streptomycin. For HMVEC-dLyAd cells, an EGMTM-2 MV Microvascular Endothelial Cell Growth Medium (Lonza, Walkersville, MD, USA) supplemented with penicillin/streptomycin was used. For starvation, primary cells were cultivated in their appropriate medium without supplementation. B16/F10 murine melanoma cell lines were obtained from ATTC (Manassas, VA, USA) and cultivated in DMEM supplemented with 10% FCS and penicillin/streptomycin.
10
Detailed Neonatal Foreskin Fibroblast Culture
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Human male neonatal foreskin primary fibroblasts (HF043, Dundee CELL products) were seeded at 8 × 103 cells/cm2 in sterile filter-cap flasks (Greiner CELLSTAR) or ~ 1000 cells per well in 384 well plates and cultured in DMEM without phenol red (Gibco 31053-028/31053-044) supplemented with 10% FBS (Biosera, FB-1001/500) and 4 mM l -Glutamine (Sigma Aldrich). All cell incubation (including SASP and viability assays, below) was conducted at 37°C in a humidified incubator at 5% CO2. No antibiotics were used; mycoplasma negative status was confirmed by regular testing by PCR (Biological Industries EZ PCR Mycoplasma test kit, Geneflow K1-0210). Cells were monitored using an EVOS digital microscope (Life Technologies) and harvested at ∼ 80% confluency using TrypLE Express (Invitrogen, 12604021). After harvesting, cells were resuspended in DMEM with FBS and 20 µL of a homogenous suspension was counted using a Cellometer T4 (Nexelcom); both cell number and cell diameter in suspension were recorded. Population doublings (PD) were calculated using the formula:
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