The esophageal cancer cell line KYSE30 (94072011; European Collection of Authenticated Cell Cultures, Salisbury, UK) was cultured in a 1:1 mixture of Ham’s F12 medium (Wako Pure Chemical Industries, Tokyo, Japan) and RPMI 1640 medium (Wako Pure Chemical Industries) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biosera, Nuaillé, France) and 1% ampicillin and streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). The human colon cancer cell line HCT116 (CCL-247; American Type Culture Collection, Manassas, VA, USA) was cultured in RPMI 1640 medium (Wako Pure Chemical Industries) supplemented with 10% FBS and 1% ampicillin and streptomycin (Thermo Fisher Scientific). Murine colon adenocarcinoma cell line MC38 (ENH204-FP; Kerafast, Boston, MA, USA) was cultured in Dulbecco’s modified Eagle medium with high glucose (Wako Pure Chemical Industries) supplemented with 10% FBS, 1 mM sodium pyruvate (Thermo Fisher Scientific), 100 μM non-essential amino acids (Thermo Fisher Scientific), 50 μg/mL gentamicin (Wako Pure Chemical Industries), 10 μM 4-(2-hydroxyethyl)-1-piperazineëthanesulfonic acid (Thermo Fisher Scientific), and 1% penicillin-streptomycin-amphotericin B (Wako Pure Chemical Industries). Cells were cultured in an atmosphere of 5% carbon dioxide (CO2) at 37°C. All experiments using cells in this study were performed for fewer than 20 passages after thawing.
Rpmi 1640 medium
Manufactured by Fujifilm
Sourced in Japan, United States, France
RPMI-1640 medium is a commonly used cell culture medium formulated to support the growth and maintenance of a wide range of cell types. It provides a balanced salt solution, essential nutrients, and other components necessary for cell cultivation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (59 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (25 mentions)
Fetal bovine serum (fbs), by Merck Group (25 mentions)
Penicillin, by Thermo Fisher Scientific (24 mentions)
Streptomycin, by Thermo Fisher Scientific (24 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (20 mentions)
Streptomycin, by Fujifilm (19 mentions)
Penicillin streptomycin, by Fujifilm (19 mentions)
Penicillin, by Fujifilm (17 mentions)
Fetal bovine serum (fbs), by Fujifilm (16 mentions)
Fetal bovine serum (fbs), by Biowest (14 mentions)
Penicillin, by Nacalai Tesque (10 mentions)
Streptomycin, by Nacalai Tesque (10 mentions)
Dulbecco s modified eagle s medium, by Fujifilm (9 mentions)
Streptomycin, by Merck Group (9 mentions)
Fetal bovine serum (fbs), by Corning (8 mentions)
Fetal bovine serum (fbs), by Biosera (8 mentions)
Fetal bovine serum (fbs), by Nichirei Biosciences (8 mentions)
Dmem medium, by Fujifilm (7 mentions)
Penicillin, by Merck Group (7 mentions)
368 protocols using rpmi 1640 medium
1
Cancer Cell Line Culture Protocols
2
Kidney Cell Phenotyping Protocol
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For cell phenotyping analysis, kidneys were harvested as described above, minced, and incubated in 10 mg/mL of Collagenase (from Clostridium histolyticum, Sigma-Aldrich, MO, USA) and 10 mg/mL of DNase (Sigma-Aldrich, MO, USA) in RPMI-1640 medium (Fujifilm Wako, Osaka, Japan) for 30 min at 37°C/5% CO2. Enzymes were inactivated by addition of 10% inactivated Fetal Bovine Serum (Biosera, Nuaillé, France)/RPMI-1640 medium (Fujifilm Wako, Osaka, Japan). The organ suspension was filtered through a 70 μm cell strainer (BD, NJ, USA), and cells were centrifuged at 700×g for 5 min. Pelleted cells were submitted to Percoll 40/75% gradient (PercollTM, GE Healthcare, Uppsala, Sweden), centrifuged at 1,500×g for 30 min, and mononuclear cells (Percoll 40/75 interphase) were collected and stained for FACS analysis. The list of antibodies used in this work is shown in online supplementary Table 1 (for all online suppl. material, see www.karger.com/doi/10.1159/000527188 ).
Data were acquired in a FACS Verse flow cytometer (8-color configuration, BD, NJ, USA) and analyzed in the software FlowJo (v.10.7.1 for Mac OS X, BD, NJ, USA). Gates were determined by the fluorescence minus one (FMO) method. Representative gating strategies for the analysis are shown in online supplementary Figure8 .
Data were acquired in a FACS Verse flow cytometer (8-color configuration, BD, NJ, USA) and analyzed in the software FlowJo (v.10.7.1 for Mac OS X, BD, NJ, USA). Gates were determined by the fluorescence minus one (FMO) method. Representative gating strategies for the analysis are shown in online supplementary Figure
3
Apoptosis and Adhesion in Podocytes
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Apoptosis assay was performed to examine the effects of serum starvation or high glucose. Human immortalized podocytes were cultured in RPMI 1640 medium (Wako) with 10% or 0.5% fetal bovine serum for 48 h for serum starvation. Human immortalized podocytes were cultured in RPMI 1640 medium (Wako) with 2000 or 5400 mg/dl for 48 h for high glucose. Then, cells were harvested for Annexin V-FITC/PI flow cytometry. Adhesion assay was performed using CytoSelect 48-well Cell Adhesion Assay (Collagen IV-coated, Colorimetric) kit (Cell Biolabs, San Diego, CA, USA). The OD 570 nm was measured in a plate reader.
4
Establishment of EBV-LCLs from Healthy Donors
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Akata (+) cells of BL origin, kindly provided by Dr. Hironori Yoshiyama [20 (link)], were cultured in RPMI1640 medium (FUJIFILM Wako Chemicals) containing 10% FCS. The productive lytic replication of Akata (+) cells was induced by treatment with anti-human IgG [21 (link)]. All cells were maintained at 37 °C in a 5% CO2 humidified atmosphere. Blood was taken (with informed consent in accordance with the Ethical Committee for Epidemiology of Hiroshima University) from EBV-seropositive healthy donors. HLA typing was performed by Wakunaga Pharmaceutical. EBV-LCLs were produced by infecting peripheral blood mononuclear cells (PBMCs) with a culture supernatant of EBV-producing Akata (+) cells in the presence of cyclosporin A (CsA). PBMCs were plated at 5 × 106 cells in a T25 culture flask in an upright position containing 2 mL of RPMI-1640 medium (FUJIFILM Wako Chemicals) supplemented with 10% FCS, 1% Penicillin-streptomycin, 200 ng/mL CsA, and 2 mL of EBV containing supernatant from Akata (+) cells. The obtained LCLs were maintained in RPMI-1640 medium supplemented with 10% FCS and 1% Penicillin-streptomycin.
5
Stromal Cell Isolation from Spleen
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For stromal cell preparation, a whole spleen was transferred into 100 μl of RPMI-1640 medium (Wako Pure Chemical Industries) and was cut and minced using scissors. After adding 900 μl of RPMI and vortexing, the spleen fragments were allowed to sediment for 1 min and the supernatant was transferred to another tube on ice. This process was repeated, collecting the supernatant each time. After collecting the hematopoietic cells, spleen capsules were digested in RPMI-1640 medium containing 4.0 mg/ml collagenase (Wako Pure Chemical Industries) with 0.1 mg/ml DNaseI (Sigma-Aldrich), and incubated for 20 min at 37 °C, with tapping every 10 min. The supernatant was transferred to another tube containing 3 ml of phosphate buffer containing 2% fetal bovine serum (FBS), 2 mM EDTA and 0.1% sodium azide on ice. This process was repeated twice, collecting the supernatant each time. The residual sediments were homogenized by using a 1 ml syringe fitted with a 27-gauge needle (Terumo). The resultant cell suspension was collected by centrifugation, as previously described23 (link).
6
Culturing Human Esophageal and Lung Cancer Cell Lines
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Human esophageal squamous cancer cell lines (TE8, TE11 and TE14) were obtained from the RIKEN BioResource Center. A human lung squamous cancer cell line (LK2) was obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). These cell lines were cultured in RPMI 1640 medium (Wako Pure Chemical Industries) supplemented with 10% FBS (Serum Source International, NC, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Nacalai Tesque, Kyoto, Japan). Establishment of GPC1 stable transfectant LK2 cell lines were described previously [4 (link)], and cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS and 250 μg/ml G418 (Life technologies, Carlsbad, CA,USA). These cell lines were cultured at 37°C under a humidified atmosphere of 5% CO2. The identity of each cell line was confirmed by DNA fingerprinting via short tandem repeat profiling, as previously described [25 (link)].
7
Phagocytic Index Determination
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At the age of 1 month, buffy coat was collected from blood samples. NH4Cl lysis solution was used to remove red blood cells. The remaining cells were rinsed with PBS, and RPMI 1640 medium (Fujifilm Wako Pure Chemical Co.) with 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA) and antibiotics (Fujifilm Wako Pure Chemical Co.) was used to suspend the cells (4.0 × 106 cells/mL). The specific conditions (37 °C for 1 h in a 5% CO2 humidified atmosphere) were adjusted to incubate the mixture of cells with 2.5% suspension of latex beads suspension labeled with FITC (1 µm diameter, L1030, Sigma-Aldrich). Ice-cold 1 mM EDTA-PBS was used to remove cell-free beads from the mixture. The remaining cells were incubated with F647-labeled anti–MHC class II (TH14B) mAbs at 200-fold dilution, and F555-labeled anti-granulocyte (CH138A, Monoclonal Antibody Center at Washington State University) mAbs at 100-fold dilution (Table S2 ), followed by analysis on a FACS CantoTM II system. The percentage of FITC+ TH14B+ cells relative to all TH14B+ cells and the percentage of FITC+ CH138A+ cells relative to all CH138A+ cells were used to determine the phagocytic index.
8
Cardiomyocyte Differentiation from hiPSCs
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Cardiomyocyte differentiation was performed using an established protocol14 (link). To maintain pluripotency, hiPSCs were cultured in dishes coated with 5 μg/cm2 fibronectin (F1141-5MG, Sigma-Aldrich) using ESF9a medium in the pre-culture stage. Cells were plated at 5 × 103 cells/cm2 one day before induction. Cardiomyocyte differentiation was co-induced by a 24-h treatment with 3 µM CHIR99021 (CHIR, 034-23103, R&D Systems) and 10 ng/mL activin A in F7 medium with insulin. Wnt/β-catenin signaling was inhibited at 1–3 days, 2–4 days, or 3–5 days after the induction of differentiation by replacing the medium with RPMI-1640 medium (189-02025, Wako) containing 2% B27 supplement lacking insulin (A18956-01, Thermo Scientific) and 5 or 10 μM IWP2 (3533, Wako, 3533/10, Nacalai tesque) to inhibit Wnt/β-catenin. The culture medium was changed every other day until day 14, and 200 μg/mL insulin was added to the medium from day 7 of the culture.
9
Culturing and Isolating Cell Lines
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To obtain cultured cells, human lymphoma cell line Daudi (JCRB9071) and Large T-transformed human embryonic kidney cell line HEK293T (RIKEN BRC2202) are cultured in D-MEM medium (Wako, 044–29765) supplemented with 10% FBS, GlutaMAX (Gibco, 25050–061), Pen/Strep (Gibco, 15140–122) or RPMI-1640 medium (Wako, 189–02025) supplemented with 10% FBS, respectively, in a humidified air condition containing 5% CO2 at 37°C. 10 μL of each con A-conjugated magnetic beads are mixed with mouse testicular cells (4×105 cells), Daudi (5×105 cells), or HEK293T (2×105 cells), respectively, and incubated for 30 min at RT. Cells bound to con A-conjugated beads are captured with a magnetic stand (FG-SSMAG2, FastGene), and numbers of con A beads-unbound cells in the supernatant are counted using a Scepter 2.0 automated cell counter (Merck) and 60 μL sensor (Fig 1C
10
Breast Cancer Cell Line Culture Protocol
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In the present study, cell lines representative of cancer types were selected as described previously (16 (link)): MCF7 and T47D for luminal A type; BT474 for luminal B type; and MDA-MB468, MDA-MB231, HCC70 and HCC1143 for TNBC. The human breast cancer cell lines were obtained from the Riken Cell Bank (MCF7; Riken BioResource Research Center, Tsukuba, Japan) and from the American Type Culture Collection (T47D, BT474, MDA-MB468, MDA-MB231, HCC70 and HCC1143; Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 100 U/ml of penicillin, 100 U/ml of streptomycin and 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified atmosphere containing 5% CO2 at 37°C.
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