HEK293T and NIH3T3 cells were gifted from Dr. Shigekazu Nagata (Osaka University). Y-79 and F9 cells were acquired from the National Institutes of Biomedical Innovation, Health and Nutrition, and RIKEN BRC, respectively. HT-1080 and SH-SY5Y cells were gifted from Dr. Michiyuki Matsuda (Kyoto University) and Dr. Yasuhisa Kimura (Kyoto University), respectively. Cells were cultured in DMEM media (Wako) with 1% Penicillin Streptomycin (PS) solution (Nacalai) and 10% Fetal Bovine Serum (FBS) (Gibco). Y-79 cells were cultured in RPMI media (Wako) with 1% PS and 20% FBS. SH-SY5Y cells were cultured in DMEM/Ham’s F-12 media (Nacalai) with 1% PS and 10% FBS. Each cell line was maintained in a 37°C, 5% CO2 incubator. For proteomics study in Figure 5 , Y-79 RD3 knockout (RD3−/−) cells and Spot-tagged RD3 expressing (RD3-Spot) cells were utilized. RD3-Spot cells were established by restoring Spot-tagged RD3 expression in RD3−/− cells. The Spot-tagged RD3 sequence was designed to delete the PAM sequence corresponding to the sgRD3 target site with the mutagenesis primer set described in Table S1 by PrimeSTAR MAX DNA polymerase (Takara).
Dmem media
Manufactured by Fujifilm
Sourced in Japan
DMEM (Dulbecco's Modified Eagle Medium) is a widely used cell culture medium formulation developed by Harry Eagle. It provides a balanced salt solution and a variety of nutrients essential for the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Primestar max dna polymerase, by Takara Bio (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
L glutamine, by Nacalai Tesque (1 mentions)
Huh7 cell line, by RIKEN Cell Bank (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
3 protocols using dmem media
1
Cell Culture Protocol for Proteomics
2
Generation of Cancer Stem Cells
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Cancer stem cells, miPS-BT549cmP cells [12 (link)] and miPS-Huh7cmP cells [14 (link)], were obtained by the conversion of miPSCs (iPS-MEF-Ng-20D-17, Lot No. 012, Riken Cell Bank, Tokyo, Japan), in which the puromycin (puro) resistant gene and green fluorescent protein (GFP) gene were cloned under the control of the Nanog promoter, in the presence of CM from human breast cancer cell line BT549 cells (ATCC HTB-122) and the human liver cancer cell line Huh7 cell line (Riken Cell Bank). All cell lines were with documents confirming their STR profiling.
CSCs were cultured using miPS medium (DMEM media (Wako, Tokyo, Japan) supplemented with 15% fetal bovine serum (FBS), 0.1 mM MEM non-essential amino acids (NEAA) (Gibco, Waltham, MA, USA), 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 50 U/mL penicillin/streptomycin, and 0.1 mM 2-mercaptoethanol (Millipore, MA, USA)) and CM from BT549 cells or Huh7 cell lines, according to the protocol designed by Said et al. [13 (link)]. The ratio of the media was 1:1 of miPS medium to CM. CSCs was maintained on 1% gelatin-coated 60 mm-dishes.
CSCs were cultured using miPS medium (DMEM media (Wako, Tokyo, Japan) supplemented with 15% fetal bovine serum (FBS), 0.1 mM MEM non-essential amino acids (NEAA) (Gibco, Waltham, MA, USA), 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 50 U/mL penicillin/streptomycin, and 0.1 mM 2-mercaptoethanol (Millipore, MA, USA)) and CM from BT549 cells or Huh7 cell lines, according to the protocol designed by Said et al. [13 (link)]. The ratio of the media was 1:1 of miPS medium to CM. CSCs was maintained on 1% gelatin-coated 60 mm-dishes.
3
Immortalized Lung and Lymph Cell Lines
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Lungs and lymph nodes were excised from the PdpnKI/KI or C57BL/6 mice, minced, and treated with collagenase/dispase. Adherent cells were infected with the supernatant from the SV40 strains 777 expressing large T antigen and cultured in DMEM media (Wako) containing 10% FBS (Sigma).
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