All cycling cells used in the study were routinely checked for possible mycoplasma contamination using EZ PCR Mycoplasma Detection Kit (Sartorius 20-700-20). Young iNets (with some residual KI67+ cells) were sporadically checked. No mycoplasma contamination was detected.
Ez pcr mycoplasma detection kit
Manufactured by Sartorius
Sourced in Israel, United States
The EZ-PCR Mycoplasma Detection Kit is a laboratory equipment product designed for the detection of mycoplasma contamination in cell cultures. It provides a rapid and reliable method for screening cell cultures for the presence of mycoplasma.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
Mm 1r, by American Type Culture Collection (2 mentions)
Mc car, by American Type Culture Collection (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
L glutamine, by Sartorius (2 mentions)
Penicillin, by Sartorius (2 mentions)
Streptomycin, by Sartorius (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Gel doctm xr, by Bio-Rad (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Jurkat, by Thermo Fisher Scientific (1 mentions)
Jurkat, by American Type Culture Collection (1 mentions)
Molt 3, by American Type Culture Collection (1 mentions)
Dnd 41, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 glutamax, by Thermo Fisher Scientific (1 mentions)
Hpb all, by Leibniz Institute DSMZ (1 mentions)
35 protocols using ez pcr mycoplasma detection kit
1
Mycoplasma Contamination Screening Protocol
2
Cell Culture of Lymphoma and Leukemia Lines
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Human diffuse large B-cell lymphoma OCI-Ly8 and B-ALL Reh cell lines were cultured and maintained in RPMI1640 medium supplemented with 10% FBS, 2 mmol/L l -glutamine, and antibiotic–antimycotic at 37°C and 5% CO2. The use of Reh cells has been described previously (29 (link)). The OCI-Ly8 cell line was a kind gift from the laboratory of Raju Chaganti (Memorial Sloan Kettering Cancer Center, New York, NY). Cell line authentication for OCI-Ly8 and Reh was performed by short tandem repeat profiling in 2021. All cell lines were routinely confirmed to be Mycoplasma free via Sartorius EZ-PCR Mycoplasma Detection Kit.
3
Mycoplasma Detection by PCR and Gel Electrophoresis
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Mycoplasma DNA was detected with a EZ-PCR Mycoplasma Detection Kit (Biological Industries, Israel) according to the manufacturer's instructions. The PCR products were then separated with 0.8% agarose gel electrophoresis and visualized under UV light (Gel DocTM XR, BIO-RAD, America).
4
Mycoplasma-free Cell Culture Validation
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The absence of mycoplasma contamination (P20) was verified with EZ-PCR Mycoplasma Detection kit (Biological Industries) according to manufacturer’s instructions. PCR products were visualized on agarose gel.
5
Generation and Characterization of Leukemia Persister Cells
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The human cell lines JURKAT (Clone E6-1) and MOLT-3 were purchased from ATCC. The human cell lines DND-41, HPB-ALL, ALL-SIL, and PEER were purchased from DSMZ. JURKAT, DND-41, and ALL-SIL were cultured in 90% RPMI-1640 Glutamax (Gibco), supplemented with 10% of heat-inactivated fetal bovine serum (FBS, Gibco) and 10,000 U/mL penicillin and streptomycin (Invitrogen). HPB-ALL and PEER were cultured in 80% RPMI-1640 Glutamax, supplemented with 20% heat-inactivated FBS and 10,000 U/mL penicillin and streptomycin. All cell lines were cultured at 37 °C, in a humidified incubator with 5% CO2, and routinely checked for mycoplasma, using the EZ PCR Mycoplasma detection Kit (Biological Industries); only mycoplasma-free cells were used. To avoid artefacts derived from long-term culture of immortalized cell lines, typically cells were not kept in culture for more than 2 months, starting from frozen stocks derived from the commercial repository.
Persister cells were generated by treating the DND-41 cells for up to 12 weeks with 0.1–1 μM GSI (GSI XXI, Compound E; Calbiochem). The GSI was chosen based on the original publication where DND-41 persister cells were generated for the first time14 (link).
Cell count was performed by trypan blue exclusion on the automated CytoSMART cell counter (Corning).
Persister cells were generated by treating the DND-41 cells for up to 12 weeks with 0.1–1 μM GSI (GSI XXI, Compound E; Calbiochem). The GSI was chosen based on the original publication where DND-41 persister cells were generated for the first time14 (link).
Cell count was performed by trypan blue exclusion on the automated CytoSMART cell counter (Corning).
6
Characterization of Leukemia and Lymphoma Cell Lines
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Cell lines used in this study included acute myeloid leukemia (AML), lymphoma and multiple myeloma. KBM3/Bu2506 or KBU, is an alkylating-agent resistant human AML cell line developed in our laboratory [23 (link)]; OCI-AML3 and MOLM13 AML cell lines were obtained from the laboratory of Dr. Michael Andreeff (UT MD Anderson Cancer Center, Houston, TX, USA). The lymphoma cell lines J45.01, Toledo, U937 and the multiple myeloma cell lines RPMI 8226, MM.1R and MC/CAR were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium (Mediatech, Manassas, VA, USA) supplemented with 10% heat-inactivated fetal bovine serum (GeminiBio, Sacramento, CA, USA) and 100 U/mL penicillin and 100 μg/mL streptomycin (Mediatech) at 37°C in a fully humidified atmosphere of 5% CO2 in air. Absence of mycoplasma contamination was confirmed using the EZ-PCR mycoplasma detection kit (Biological Industries, Cromwell, CT, USA).
7
Gastric Cancer Tissue Sourcing and Cell Line Characterization
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Biopsy specimens of non-tumorous gastric mucosa were obtained from the gastric antrum of four Japanese GC patients and three healthy Japanese individuals who underwent upper gastrointestinal endoscopy at Sapporo Medical University Hospital. This study was approved by the institutional review board of Sapporo Medical University (No. 29-18). Informed consent was obtained from all participants prior to specimen collection. For the chromatin immunoprecipitation-sequencing (ChIP-seq) experiments, crypt isolation was performed to obtain an epithelial cell-enriched fraction [14 (link)]. For targeted sequencing, biopsy specimens of paired tumor and non-tumorous gastric mucosa were obtained from eight Japanese patients with GC. Gastric cancer cell lines (HSC-45, MKN45, SNU1, and SNU638) were cultured as previously described [15 (link), 16 (link)]. Cell lines were authenticated using short tandem repeat analysis (JCRB, Tokyo, Japan; BEX, Tokyo, Japan). Mycoplasma infection was negative in the above cell lines, as confirmed using the EZ-PCR Mycoplasma Detection Kit (Biological Industries, Beit HaEmek, Israel).
8
Cultivation of Murine Astrocytoma Cell Line
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The murine astrocytoma cell line, ALTS1C1 (BCRC60582, BCRC, Hsinchu, Taiwan; T8239, Applied Biological Materials, BC, Canada), was previously established by our laboratory [38 (link)]. ALTS1C1 cells were incubated at 37 °C/5% CO2 humidified air condition and maintained in the culture medium. Culture medium was prepared in Dulbecco’s modified Eagle’s medium (DMEM; Gibco®, 12100046, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; Gibco®, 16000044) and 1% penicillin-streptomycin (PS; Gibco®, 15140122). Mycoplasma contamination was examined by a EZ-PCR™ Mycoplasma Detection Kit (Biological Industries, 20-700-20, Beit Haemek, Israel) before use.
9
Cell Line Maintenance and Validation
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The MOLM-13 cell line was purchased from DSMZ line (catalog no. ACC-554, RRID: CVCL_2119), while the MV-4-11 and U-2 osteosarcoma (OS) cell lines were purchased from ATCC (catalog no. CRL-9591, RRID: CVCL_0064; catalog no. HTB-96, RRID: CVCL_0042). The HB11;19 cell line (RRID: CVCL_8227) was kindly provided by Dr. Yana Pikman. MOLM-13 and HB11;19 cells were cultured in RPMI1640 with GlutaMAX (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin (P-S). MV-4-11 cells were cultured in Iscove's Modified Dulbecco's Medium with GlutaMAX (Gibco) supplemented with 25 mmol/L HEPES (Gibco), 10% FBS, and 1% P-S. The human osteosarcoma cell line U-2 OS was cultured in DMEM with GlutaMAX (Gibco) supplemented with 10% FBS and 1% P-S. AML cell lines were starved in respective serum-free media, supplemented with 1% BSA (Sigma-Aldrich). Cell lines were kept in a humidified incubator at 37°C, with 5% CO2. All cell lines were regularly checked for Mycoplasma using the EZ PCR Mycoplasma detection Kit (Biological Industries). To avoid artefacts of long-term culture of immortalized cell lines, cell cultures were started up from an early passage vial and interrupted after 8–12 weeks.
10
Cell Culture Protocol for HEK293T, COS7, and HCT116
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HEK293T (internal stock) and COS7 (internal stock) cells were cultured in complete DMEM (Biological Industries, Israel), and HCT116 (kind gift from Prof. Moshe Oren, Weizmann Institute, Israel) cells were cultured in McCoy’s 5A medium (Biological Industries). Culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin G sodium, and 0.1 mg/mL streptomycin sulfate. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. All cell lines were confirmed to be mycoplasma-free using the EZ-PCR Mycoplasma Detection Kit (Biological Industries).
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