Four GBM cell lines were employed in several combinations in order to simulate cell population heterogeneity as one might encounter in GBM tumors. GBM cell lines U-373 MG (ATCC® HTB-16TM) U-87 MG (ATCC® HTB-14TM), U-87 EGFRvIII cell line (gifted by Dr. Webster Cavenee from Ludwig Cancer Research Institute, San Diego) and A172 (ATCC® CRL-1620TM) (obtained from ATCC, Manassas, VA) were employed. All the cancer cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) - high glucose with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin in a tissue culture incubator at 37°C with 5% CO2. In addition, human astrocytes (Sciencell Research Laboratories, Inc., Carlsbad, CA) were cultured in Astrocyte Medium containing 2% FBS, 1% astrocyte growth supplement, and 1% penicillin/streptomycin at 37°C with 5% CO2. Cells were recovered from tissue culture plastic for subsequent studies using Trypsin/EDTA (Thermo Fisher, Waltham, MA).
Htb 14tm
Manufactured by American Type Culture Collection
Sourced in Germany, United States
The HTB-14TM is a laboratory equipment product offered by American Type Culture Collection. It is designed for cell culture applications. The core function of this product is to provide a controlled environment for the growth and maintenance of cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
U87mg, by American Type Culture Collection (2 mentions)
Human astrocyte, by ScienCell (2 mentions)
Ln229, by American Type Culture Collection (2 mentions)
U373mg, by American Type Culture Collection (1 mentions)
Antibiotics antimycotic, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Oncor, by Siemens (1 mentions)
Svg p12, by American Type Culture Collection (1 mentions)
6 protocols using htb 14tm
1
Establishing Glioblastoma Cell Heterogeneity
2
Culturing U87-MG Glioblastoma Cells
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The integrin αvβ3 positive human glioblastoma cell, Uppsala87-Malignant Glioma (U87-MG, American Type Culture Collection (ATCC)® HTB-14TM; ATCC-LGC Standard, Wesel, Germany) was cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics/antimycotics (Gibco, Grand Island, NY, USA) at 37 °C in 5% CO2.
3
Cell Irradiation and Culturing Protocol
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U87 and LN229 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; U87: ATCC® HTB-14TM; LN229: ATCC® CRL-2611TM). Both cell lines were cultured as described elsewhere [24 (link)]. 24 h prior to the start of experiments, the culture medium was changed and the cells were irradiated with 2 Gy, with a dose rate of 2 Gy/min using 6 MV photons (Siemens, Erlangen, Germany, Siemens ONCOR).
4
Characterization of Human Glioblastoma Cell Lines
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The human GBM cell lines U87MG (ATCC, HTB-14 TM), U87MGwtEGFR (stably overexpressing wtEGFR) [62 (link)], and LN229wtEGFR (stably overexpressing wtEGFR) have been described [43 (link)]. Expression of wtEGFR was verified by Western blot. Human astrocytes (ScienCell Research Laboratories) and neural human progenitor cells (NHPC) (Lonza) were grown as recommended by suppliers. Normal human adult brain tissue, obtained from patients undergoing epilepsy surgery at Emory University (IRB protocol 642-2005), was cultured in DMEM/F12 (50/50 mix, Cellgro) and 10% FCS (HyClone) in the presence of sodium pyruvate, Penstrep, and L-glutamine (HyClone). Cell lines were used for fewer than 6 months after resuscitation and were routinely tested for Mycoplasma, no genotypic authentication was conducted. Each cell line was used in early passage.
5
Systematic Review of TMZ Effects on U-87 MG Cells
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Systematic literature search and screening
The systematic search was conducted on the databases PubMed, Embase and Web of Science on the 26 th of August 2020 using the search strategy described in Supplement 1. Two reviewers independently screened article titles and abstracts for potential inclusion, with discrepancies resolved by a third reviewer. This was followed by full text screening. We included studies describing controlled in vitro cell culture experiments that compared the effect of a single TMZ treatment on the viability of U-87 MG cells with that in untreated controls. We also required that cell viability was measured by colorimetric assay or by cell counting; and that the authors used Dulbecco's Modified Eagle Medium (DMEM) as the ATCC recommends using a modified Eagle medium for U-87 MG cell cultures (HTB-14 TM , ATCC®, 2021). We only included original peer-reviewed research articles in the English language, with no restrictions made on publication year. Our protocol had included consideration of cell growth rates in xenotransplantation models, but we later decided to focus exclusively on in vitro research. Inclusion and exclusion criteria are given in Supplement 2.
The systematic search was conducted on the databases PubMed, Embase and Web of Science on the 26 th of August 2020 using the search strategy described in Supplement 1. Two reviewers independently screened article titles and abstracts for potential inclusion, with discrepancies resolved by a third reviewer. This was followed by full text screening. We included studies describing controlled in vitro cell culture experiments that compared the effect of a single TMZ treatment on the viability of U-87 MG cells with that in untreated controls. We also required that cell viability was measured by colorimetric assay or by cell counting; and that the authors used Dulbecco's Modified Eagle Medium (DMEM) as the ATCC recommends using a modified Eagle medium for U-87 MG cell cultures (HTB-14 TM , ATCC®, 2021). We only included original peer-reviewed research articles in the English language, with no restrictions made on publication year. Our protocol had included consideration of cell growth rates in xenotransplantation models, but we later decided to focus exclusively on in vitro research. Inclusion and exclusion criteria are given in Supplement 2.
6
GSK-3 Inhibitor IX Effects on Glioblastoma and Fetal Glial Cells
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Group 1: Glioblastoma multiforme (GBM) [U-87MG (ATCC HTB-14 TM)] Group 2: Human fetal glial cell line [SVGp12 (ATCC CRL-8621)] Group 3: GBM + GSK-3 (glycogen synthase kinase) inhibitor Group 4: Human fetal glial cell line + GSK-3 (glycogen synthase kinase) inhibitor These experimental groups were incubated for 24, 48, and 72 hours following administration of 0.5-, 1-, 1.5-, 2-, and 2.5-μM doses of GSK-3 inhibitor IX to examine its IX effects. The effects of GSK-3 inhibitor IX on cell viability (apoptosis) were investigated.
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