The DT40 cell line was derived from chicken B lymphoma [30 (link)] and was cultured in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated FBS (fetal bovine serum), 1% chicken-serum, 50 μM mercaptoethanol (Nacalai Tesque), L-glutamine (Nacalai Tesque), 50 U/ml penicillin, and 50 μg/ml streptomycin (Nacalai Tesque). The cell line was maintained at 39.5°C in a humidified atmosphere and 5% CO2. TK6 cell line is a human B lymphoblastoid line [31 (link)] and cultured in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 5% heat-inactivated horse-serum, L-glutamine (Nacalai Tesque), 0.2 mg/ml Sodium pyruvate (Sigma-Aldrich), 50 U/ml penicillin, and 50 μg/ml streptomycin (Nacalai Tesque). The TK6 cells were maintained at 37°C in a humidified atmosphere and 5% CO2. The list of the mutant clones we analyzed in this manuscript is shown in Table 1 .
Rpmi 1640 medium
Manufactured by Nacalai Tesque
Sourced in Japan, United States, United Kingdom, Germany
RPMI 1640 medium is a commonly used cell culture medium formulated to support the growth of a variety of cell types, including human and animal cells. It is a balanced salt solution that provides essential nutrients, vitamins, and amino acids required for cell maintenance and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (100 mentions)
Penicillin, by Nacalai Tesque (65 mentions)
Streptomycin, by Nacalai Tesque (64 mentions)
Amphotericin b, by Nacalai Tesque (37 mentions)
Cho k1, by American Type Culture Collection (21 mentions)
Fetal bovine serum (fbs), by Biowest (20 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (18 mentions)
Penicillin, by Thermo Fisher Scientific (17 mentions)
Streptomycin, by Thermo Fisher Scientific (17 mentions)
Penicillin streptomycin, by Nacalai Tesque (14 mentions)
Dulbecco s modified eagle s medium dmem, by Nacalai Tesque (13 mentions)
Fetal bovine serum (fbs), by Nacalai Tesque (12 mentions)
Fetal bovine serum (fbs), by Equitech-Bio (12 mentions)
Fetal bovine serum (fbs), by Merck Group (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (12 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (11 mentions)
Cho k1, by Nacalai Tesque (11 mentions)
Neon transfection system, by Thermo Fisher Scientific (8 mentions)
Ln229, by American Type Culture Collection (8 mentions)
Dulbecco s modified eagle s medium, by Nacalai Tesque (8 mentions)
269 protocols using rpmi 1640 medium
1
Culturing B Lymphoma and B Lymphoblastoid Cell Lines
2
Culturing Various Cell Lines
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The HL-60 cells used here were donated by the National Hospital Organization, Osaka Minami Medical Center (Osaka, Japan). HL-60 cells were grown in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA). The human breast cancer cell line MDA-MB-231 (92020424) was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). The human gastric cancer cell line KATO III (JCRB0611) was obtained from the Health Science Research Resources Bank (HSRRB, Osaka, Japan). The human glioblastoma multiforme cell line T98G (RCB1954) was provided by the RIKEN BRC through the National Bio-Resource Project of MEXT (Ibaraki, Japan). These cell types were grown in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum. Cells were incubated in a CO2 incubator (MCO-19AIC, SANYO, Osaka, Japan) at 37 °C in a humidified atmosphere containing 5% CO2.
3
Establishment of Mesothelioma Cell Lines
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Human mesothelioma H2052 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured at 37℃ under 5% CO2 in Dulbecco's modified Eagle's medium or RPMI medium 1640 supplemented with 10% heatinactivated fetal bovine serum and antibiotics (Nacalai Tesque, Kyoto, Japan). Mitochondrial DNA-depleted ρ0 cells were established through long-term treatment of H2052 cells with ethidium bromide (25 ng/ml), as described previously (King and Attardi, 1989). Cisplatin was purchased from Sigma-Aldrich (St. Louis, MO). Other reagents used were of the highest grade commercially available.
4
Isolation of Murine Bone Marrow Cells
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The nucleated bone marrow cells were collected according to [29 (link)]. In brief, mice at eight postnatal weeks were euthanized by CO2 asphyxiation. Both femurs of each mouse were removed, and the bone marrow tissues were collected by flushing femurs with phosphate-buffered saline free from calcium and magnesium ions. After treating bone marrow tissues with tris-buffered ammonium chloride for the lysis of erythrocytes and washing with RPMI medium 1640 (Cat. 06261-65, with L-glutamine and without phenol red, Nacalai Tesque, Inc., Tokyo, Japan), single cell suspensions of dissociated nucleated bone marrow cells were filtered through a 40 μm cell strainer (Corning, Inc., New York, NY, USA) and then counted for further use.
5
Culturing and Harvesting HCT 116 Colorectal Cancer Cells
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Colorectal cancer cell line, HCT 116, was cultured in Roswell Memorial Institute (RPMI) medium 1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Tico Europe Ltd., Amstelveen, Netherlands), 1% Antibiotic-Antimycotic solution (Nacalai Tesque, Kyoto, Japan), maintained at 37 °C in a humidified 5% CO2-enriched atmosphere (ESCO, Horsham, PA, USA). Cells were rinsed by using 1× phosphate-buffered saline (PBS) (Nacalai Tesque, Kyoto, Japan), repeated three times, and cells were detached by using trypsin-EDTA (Life Technologies, Burlington, Canada), followed by RPMI addition to stop the trypsinization; cells were pelleted by centrifugation at 1000 rpm, resuspended in RPMI for sub-culturing or seeding purpose, or 1× PBS was used to resuspend for harvesting as a washing step, followed by second-time pelleting of the cells. During harvesting, after observation under the microscope and cell count was conducted, cells from four flasks were pooled and pelleted in the amount as required for further analyses. The microcentrifuge tubes containing a pelleted known number of cells were immediately flash frozen in liquid nitrogen before storage in −80 °C until further usage.
6
Cell culture conditions for various cell lines
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HEK293 cells (ATCC CRL-1573), HT-29 cells (ATCC HTB-38), U-251 MG cells (CVCL_0021), SH-SY5Y cells (ATCC CRL-2266), HeLa cells (ATCC CCL-2), and their KO derivatives were cultured in Dulbecco's Modified Eagle Medium (DMEM) with high glucose (Nacalai Tesque). Raji cells (ATCC CCL-86), THP-1 cells (ATCC TIB-202), K-562 cells (ATCC CCL-243), and their derivatives were cultured in RPMI medium 1640 (Nacalai Tesque). HAP1 cells (kindly provided by Thijn Brummelkamp, The Netherlands Cancer Institute, Amsterdam, The Netherlands) and their KO derivatives were cultured in Iscove's Modified Dulbecco's Medium (Nacalai Tesque). CHO (Chinese Hamster Ovary) K1 cells (ATCC CCL-61) and their derivatives were maintained in DMEM/F-12 (Nacalai Tesque). All cell culture medium contained 10% heat-inactivated fetal bovine serum (FBS) (172012-500ML, Sigma). All cells were maintained in an incubator at 37 °C and 5% CO2.
7
Ultrastructural Analysis of K562 Cells
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K562 cells, which are cell lines originated from human chronic myelogenous leukemia, were cultured in suspension at 37 C in RPMI-1640 medium (Nacalai Tesque, Kyoto) supplemented with 10% FBS (Life Technologies, Grand Island, NY) plus antibodies (Nacalai Tesque) (Hirao et al. 2018) (link).
For CF-FS, glutaraldehyde was added directly to the cell suspension to a final concentration of 2.5%. They were kept at 37 C for 10 min and stored at 4 C overnight. They were collected by centrifugation at 1400 rpm (400 G, swing rotor) for 7 min, and sandwiched between two copper discs (Fig. 1c). Likewise, for RF-FS of living cultured cells, cells were collected by centrifugation at 1400 rpm for 7 min and sandwiched between two copper discs.
The sandwiched cells were snap-frozen by plunging into melting propane cooled with liquid nitrogen. They were freeze substituted in acetone containing 2% osmium tetroxide for 2-4 days at 80 C and embedded in epoxy resin. Ultrathin sections were cut to a thickness of 50 nm with a diamond knife, picked up on single-slot grids, and placed on a Formvar film mounted on an aluminum rack (Yamaguchi and Chibana 2018) . They were stained with uranyl acetate and lead citrate (Yamaguchi et al. 2005b) , and observed in a JEM-1400 electron microscope (JEOL, Tokyo) at 100 kV as with human tissues (Fig. 1) (Yamaguchi and Chibana 2018) .
For CF-FS, glutaraldehyde was added directly to the cell suspension to a final concentration of 2.5%. They were kept at 37 C for 10 min and stored at 4 C overnight. They were collected by centrifugation at 1400 rpm (400 G, swing rotor) for 7 min, and sandwiched between two copper discs (Fig. 1c). Likewise, for RF-FS of living cultured cells, cells were collected by centrifugation at 1400 rpm for 7 min and sandwiched between two copper discs.
The sandwiched cells were snap-frozen by plunging into melting propane cooled with liquid nitrogen. They were freeze substituted in acetone containing 2% osmium tetroxide for 2-4 days at 80 C and embedded in epoxy resin. Ultrathin sections were cut to a thickness of 50 nm with a diamond knife, picked up on single-slot grids, and placed on a Formvar film mounted on an aluminum rack (Yamaguchi and Chibana 2018) . They were stained with uranyl acetate and lead citrate (Yamaguchi et al. 2005b) , and observed in a JEM-1400 electron microscope (JEOL, Tokyo) at 100 kV as with human tissues (Fig. 1) (Yamaguchi and Chibana 2018) .
8
CCL2-induced MUC5AC and SLC26A4 expression
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NCI-H292 cells were obtained from ECACC (Porton Down, UK) and cultured in RPMI-1640 medium (Nacalai Tesque) containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C. Cells at 80% confluency were treated with or without recombinant human CCL2 (100 ng/ml, Peprotech, Rocky Hill, NJ, USA) for 18 h at 37 °C. qPCR was performed with the following primer sets: MUC5AC, 5′-tggggacagctcttccatgtac-3′ and 5′-tgcagtgcagggtcacattc-3′; SLC26A4, 5′-tttcctggacgttgttggagtg-3′ and 5′-tgtcgtcaaagaacccgcattg-3′; GAPDH, 5′-tgcaccaccaactgcttagc-3′ and 5′-atggcatggactgtggtcatg-3′.
9
Isolation and culture of primary hepatocytes and macrophages
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Primary hepatocytes were obtained from miR‐33 knockout mice by the collagenase perfusion method. Livers from mice were lysed by retrograde perfusion with collagenase type II (Worthington, OH). The cell suspension was passed through a 70‐μm cell strainer and centrifuged to isolate mature hepatocytes. Hepatocytes were seeded onto collagen type‐I coated dishes at a density of 2.5×105 cells/mL in Dulbecco's modified Eagle's medium low glucose (Nacalai Tesque, Japan) supplemented with 10% fetal bovine serum (FBS). Primary mouse peritoneal macrophages were obtained from miR‐33 WT, miR‐33a knockout, miR‐33b KI, and miR‐33 KOKI mice by intraperitoneal lavage with cold phosphate‐buffered saline (PBS) 72 hours after the intraperitoneal administration of 3% (w/v) thioglycolate. Macrophages were resuspended in RPMI 1640 medium (Nacalai Tesque) containing 10% FBS, and seeded at a density of 5.0×105 cells/mL. Hep‐G2 cells were cultured with DMEM supplemented with 10% FBS at a density of 1.0×106 cells/mL. For cholesterol‐depletion experiments, the medium was replaced with DMEM containing 10% lipoprotein‐depleted serum and 10 nmol/L pitavastatin.
10
Culturing Human Gastric Cancer Cells
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The poorly differentiated human gastric adenocarcinoma cell line MKN45 was used in the present study. Cells were grown in plastic culture flasks (Corning Incorporated, NY, USA) and maintained in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin. The flasks were kept in a humidified incubator at 37°C under 5.0% CO2 in air.
The 140 mM isotonic NaCl solution (NaCl buffer) contained 140 mM NaCl, 5.0 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 5.0 mM glucose, and 10 mM HEPES.
The 140 mM isotonic NaCl solution (NaCl buffer) contained 140 mM NaCl, 5.0 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 5.0 mM glucose, and 10 mM HEPES.
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