Wild-type Listeria monocytogenes stably expressing the MHC class I-restricted epitope HSV gB498-505 (gB-8p), designated LM-gB (28 (link)), was generously provided by Dr. Sing Sing Way (Cincinnati Children's Hospital, OH). Prior to infection, Lm-gB was freshly plated on BHI plates containing streptomycin selection antibiotic. Colonies were selected from the plate for growth in liquid culture. Bacteria were grown to log phase and mice were injected intravenously (i.v.) with either 5 × 103 colony forming units (CFU) in 100 ul of PBS for naïve mice (~0.1 LD50) and 5 × 104 CFU for immune mice to demonstrate protective immunity. Recombinant vaccinia virus expressing the MHC class I- restricted epitope HSV gB498-505 (gB-8p), designated VACV-gB (25 (link)), was generously provided by Dr. S.S. Tevethia (Pennsylvania Sate University, College of Medicine). VACV-gB viral stocks were propagated and quantified in 143B cells (ATCC). Mice were infected with 2 × 105 plaque forming units (PFU).
143b cells
Manufactured by American Type Culture Collection
Sourced in United States
143B cells are a human osteosarcoma cell line derived from a bone tumor. They serve as a model system for the study of bone biology and related disease processes.
Automatically generated - may contain errors
Lab products found in correlation
Nih3t3 cells, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Advanced dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Hos cells, by American Type Culture Collection (1 mentions)
Viafect, by Promega (1 mentions)
U2os cell, by American Type Culture Collection (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
U2os htb 96, by American Type Culture Collection (1 mentions)
Nci h2122, by American Type Culture Collection (1 mentions)
Rpmi 1640 media, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Merck Group (1 mentions)
Eagle s minimum essential medium, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Bovogen (1 mentions)
Mc57g cells, by American Type Culture Collection (1 mentions)
Hek293t, by National Collection of Authenticated Cell Cultures (1 mentions)
12 protocols using 143b cells
1
Listeria monocytogenes-based Vaccine Evaluation
2
Murine Osteosarcoma Cell Lines
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The highly aggressive murine OS cell line LM8 was obtained from RIKEN BRC (Ibaraki, Japan). NIH/3T3 cells, HOS cells (non-aggressive, wild-type k-Ras, human OS), and 143B cells (highly aggressive; k-Ras activated, human OS) (20 (link), 21 (link)) were obtained from the ATCC (Manassas, VA, USA). These cells were cultured in Advanced DMEM (Thermo Fisher Scientific, Waltham, MA, USA) with 2% heat-inactivated FBS (Bio-West, Nuaillé, France). LM8-WT or LM8 TSG101-KO cells were generated by the CRISPR–Cas9 system. The pX330Puro or pX330Puro-TSG101 plasmid was transfected into LM8 cells using ViaFect (Promega, Madison, WI, USA). LM8 TSG101-KO cells were monocloned, resulting in the generation of two monoclonal LM8 TSG101-KO cell lines (KO1 and KO2). LM8-WT cells were maintained as a polyclonal population.
3
Culturing NIH3T3, 143B, and U2OS Cells
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NIH3T3 cells (ATCC), 143B cells (ATCC) and U2OS cells (ATCC) were cultured in DMEM medium (GIBCO) with 10% FBS (GIBCO) and 5% CO2 at 37 °C.
4
Culturing 143B cells with/without mtDNA
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143B cells were from ATCC (Manassas, VA, USA, Cat# CRL-8303). All cells were propagated in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum, 50 µg/mL gentamycin, 50 µg/mL uridine, and 1 mM sodium pyruvate in a humidified atmosphere containing 5% CO2 at 37 °C. This medium is permissive for the growth of cells devoid of mtDNA (ρ0 cells; +UP medium). When indicated, uridine and pyruvate were omitted from this medium for selection against ρ0 cells (−UP medium).
5
Culturing 143B Cells with Supplements
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143B cells were purchased from ATCC and cultured in MEM GlutaMAX with 1 × MEM non-essential amino acids, 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin.
6
Generation of Isogenic Sarcoma Cell Lines
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To generate Atrx isogenic mouse sarcoma cell lines, pSP Cas9(BB)-2A-GFP (PX458) with Atrx gRNA or vector control were transfected into newly derived primary soft tissue sarcoma cell lines (KP) using the TransIT-LT1 transfection reagent (Mirus). Cell lines from transfected single-cell clones were then screened for loss of ATRX protein expression by Western blot. Antibodies used for Western blot and immunofluorescence are listed in Supplemental Table 1 . To generate human sarcoma cell lines, 143B cells were purchased (ATCC, CRL8303) and an identical procedure was performed, except the gRNA in the PX458 vector was targeted to the human ATRX gene. Passaging of sarcoma cell lines was performed in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 1% Pen-Strep (Thermo Fisher Scientific), and 1% L-glutamine (Thermo Fisher Scientific). Cell lines were incubated at 37°C with 5% CO2 in a humidified cell culture incubator. U2OS (HTB-96) and 143B (CRL-8303) cell lines were purchased from ATCC.
7
Cell Line Protocols for PRISM Screening
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Cell lines used in PRISM screening have been previously described33 (link),34 (link). 143B cells were obtained from ATCC and cultured in Eagle’s MEM with 0.015 mg/mL 5-bromo-2’-deoxyuridine and 10% FBS. NCIH650 and NCIH2122 cells were obtained from ATCC and cultured in RPMI with 10% FBS. Kelly cells were obtained from DSMZ and cultured in RPMI with 10% FBS. RhJT and TE617T cells were a gift of the Broad Institute Pediatric Dependencies Project and cultured in RPMI with 10% FBS. HDMB03 and MB002 cells were generously provided by Till Milde (KiTZ, Heidelberg) and Yoon-Jae Cho (OHSU), respectively, and cultured in neurosphere medium as previously described49 (link). All cells were validated to be free of mycoplasma spp. with routine testing for identity by short-tandem repeat testing.
8
Cell Culture Maintenance for Viral Assays
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The MCL22 was maintained in CD‐CHO media supplemented with 8 mm l ‐glutamine, penicillin–streptomycin (1 U mL−1 and 0.1 mg mL−1, respectively) solution, hygromycin B (500 μg mL−1) and puromycin (10 μg mL−1). 143B cells (ATCC CRL‐8303; ATCC, Virginia, USA) were maintained in RPMI‐1640 media (Sigma Aldrich, MO, USA) supplemented with 10% fetal bovine serum (Bovogen, VIC, Australia), 2 mm l ‐glutamine and penicillin–streptomycin. Virus titration was performed in 143B cells expressing D13 (ST01‐33) maintained in 143B culture media supplemented with hygromycin B (500 μg mL−1). MC57G cells (ATCC CRL‐2295; ATCC, Virginia, USA) were maintained in Eagle’s Minimum Essential Medium (Sigma Aldrich, MO, USA) supplemented with 10% fetal bovine serum, 2 mm l ‐glutamine and penicillin–streptomycin. ACE2‐HEK293 recombinant cell line (catalog number 79951; BPS Bioscience, San Diego, CA, USA) was maintained in Dulbecco’s Modified Eagle Medium supplemented (Sigma Aldrich, MO, USA) with 10% fetal bovine serum, 2 mM l ‐glutamine and penicillin–streptomycin. All cell culture reagents were purchased from Thermo Fisher Scientific, Waltham, MA, USA, unless otherwise specified.
9
Cell Culture and Plasmid Construction
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Saos‐2, U2OS, MG63, Fhob1.19, L6, and HEK293T cells were purchased from the cell bank of the Chinese Academy of Science (Shanghai, China). 143B cells were purchased from the American Type Culture Collection (ATCC). Except for the Saos‐2 cells that were cultured in McCoy's 5A Medium (Gibco, USA), all other cells were cultured in Dulbecco's modified Eagle medium (DMEM) (HyClone, USA) supplemented with 10% fetal bovine serum (FBS; VWR, USA) and 100 U mL−1 penicillin/streptomycin (Gibco, USA). All cell lines were maintained in an incubator (Thermo Fisher Scientific, USA) with 5% CO2 at 37 °C. Cycloheximide, DOX, TBHq, and brusatol were purchased from Selleck (Shanghai, China). KI696 was purchased from MedChemExpress (China). The DDRGK1 shRNA and sgRNA plasmids, DDRGK1‐flag plasmid, DDRGK1‐Myc‐plasmid, NRF2‐HA plasmid, KEAP1‐HA plasmid, KEAP1‐HA truncation plasmid (amino acids 1–320321‐644), DDRGK1‐Flag truncation plasmid (amino acids 1–210, 210–280, 280–314), and Cas9 lentivirus were constructed by IBSBio (Shanghai, China).
10
Listeria monocytogenes Infection Model in Mice
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Mice were i.v. infected with 5 × 103 CFU of either wild-type Listeria monocytogenes [strain 10403, obtained from Dr. Nikolich-Zugich (18 (link)), designated WT-LM], or a recombinant strain of L. monocytogenes expressing the gB peptide, designated LM-gB [obtained from Dr. S. Sing Way (19 (link))]. Unless stated otherwise, mice were inoculated with 5 × 103 CFU of LM-gB. For all experiments, colonies were selected from the plate for growth in liquid culture. Bacteria were grown to log phase, and mice were injected i.v. Recombinant vaccinia virus expressing the gB peptide (VACV-gB) (20 (link)) was generously provided by Dr. S.S. Tevethia (Pennsylvania State University, College of Medicine). VACV-gB viral stocks were propagated and quantified in 143B cells (American Type Culture Collection), as previously described (21 (link)). Mice were infected with 2 × 105 PFU of VACV-gB (i.p.).
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