CRC cell lines (5–10 × 103 cells/well) were seeded in wells of Nunc F96 Microwell white plates (Thermo Fisher Scientific) in a final volume of 100 µL culture medium containing 200 µg/mL D-luciferin (Promega) per well. After over-night incubation, luminescence was scored using a GloMax-Multi detection system (Promega). This initial measurement was defined as the day 0-value; luminescence was then determined daily. The cell proliferation rate for each cell line was estimated as the ratio of photon count at the chosen timepoint to that on day 0 [36 (link)]. For the cytotoxicity assay, CRC cell lines (5–10 × 103 cells/well) were seeded in 96-well white cell culture plates. After over-night incubation, irinotecan (CPT-11; Wako), oxaliplatin (L-OHP; Wako) or 5-fluorouracil (5-FU; Wako) were added at the indicated concentration and luminescence was determined at 24, 48, 72 h after administration of anti-cancer drugs. Cell viability was calculated as the treated/control cell ratio of photon counts.
5 fluorouracil 5 fu
Manufactured by Fujifilm
Sourced in Japan
5-fluorouracil (5-FU) is a synthetic pyrimidine analog that inhibits the synthesis of DNA and RNA. It is a commonly used chemotherapeutic agent in the treatment of various types of cancer.
Automatically generated - may contain errors
Lab products found in correlation
D luciferin, by Promega (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
Cell counting kit 8 (cck8), by Fujifilm (1 mentions)
Cell counting kit 8 (cck8), by Corning (1 mentions)
Human dermal fibroblasts, by PromoCell (1 mentions)
Cisplatin, by Fujifilm (1 mentions)
Hesperidin, by Fujifilm (1 mentions)
Pd0332991, by Merck Group (1 mentions)
Sb202190, by Cayman Chemical (1 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
E cadherin, by R&D Systems (1 mentions)
α smooth muscle actin, by Agilent Technologies (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse egf, by Thermo Fisher Scientific (1 mentions)
A83 01, by Adooq (1 mentions)
Primocin, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Cayman Chemical (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
12 protocols using 5 fluorouracil 5 fu
1
Colorectal Cancer Cell Viability Assay
2
Evaluating Induced Pluripotent Cell Toxicity
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Cell toxicity of generated IPCs was investigated using the Cell Counting kit-8 (Fuji film-Wako, Osaka, Japan). Briefly, we incubated human dermal fibroblasts (Promo Cell, Heidelberg, Germany) in 24-well plates for 24 h at 37 °C after cell count. After 24 h co-culture5 (link) at 37 °C with three IPCs per well (a transwell system, 0.4 μm pore size membrane; Corning, Acton, MA), the solution of Cell Counting kit-8 was added. As negative control, we added 100 μM 5-Fluorouracil (5-FU, Wako) to fibroblast. Then, the proliferation of fibroblasts was analysed by enzyme-linked immunosorbent assay (AKRIN-011H, Shibayagi, Shibukawa, Japan) with a microplate reader at a wavelength of 450 nm.
3
Evaluation of Cytotoxicity on EATC and 3T3-L1 Cells
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The proliferation and viability of EATC and 3T3-L1 cells were evaluated using a trypan blue assay. EATCs (1.0 × 106 cells/ml/φ 45-mm dish) were cultured with or without KGE (0−50 μg/ml), EMC (0−400 μM; M1204, purity >98.0%, Tokyo Kasei Kogyo Co., Tokyo, Japan), hesperidin (400 μM; 088–0734, FUJIFILM Wako Pure Chemical, Osaka, Japan), 5-fluorouracil (5-FU; 100 μM; 068–01401, FUJIFILM Wako Pure Chemical), cisplatin (6.25 μM; 033–20091, FUJIFILM Wako Pure Chemical) or PD0332991 (20 μM; PZ0199, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. EATCs were collected and subsequently stained with trypan blue (0.4%). 3T3-L1 cells (1.0 × 105 cells/ml/φ 35-mm dish) were cultured with or without KGE (0−200 μg/ml) and EMC (0−800 μM) for 24 h 3T3-L1 cells were collected and subsequently stained with trypan blue (0.4%). Viable and dead cells were counted under a microscope. The cell viability was calculated using the following formula: % cell viability = [(total cells – dead cells)/total cells] × 100. The 50% lethal concentration (LC50) was determined by regression analysis of cell viability data. The selectivity index (SI) was calculated using the following formula: Selectivity index (SI) = LC50 of 3T3-L1 cells (normal cells)/LC50 of EATC.
4
Organoid Generation from Colorectal Tissues
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To generate organoids, human colorectal tissues were cultured with modified ISCs media as described previously [9 (link)]. The components were as follows: Advanced Dulbecco's Modified Eagle's Medium (DMEM) with 50% Wnt, Noggin, and R-Spondin conditioned medium; GlutaMax; B-27 supplement; 100 μg/mL Primocin (Invitrogen, Carlsbad, CA, USA); 1 mM N-acetyl-L-cysteine; 10 mM Nicotinamide (Sigma-Aldrich, St. Louis, MO, USA); 50 ng/mL mouse EGF (PeproTech, Inc., Rocky Hill, NJ, USA); 500 nM A83-01 (Adooq Bioscience, Irvine, CA, USA); 3 μM SB202190; 10 μM Y-27632 (Cayman, Ann Arbor, MI, USA). Anticancer drugs were as follows: 5-fluorouracil (5-FU) (WAKO, Tokyo, Japan); Irinotecan (LC Laboratories, Woburn, MA, USA). Antibody sources were as follows: E-cadherin (R&D System, Minneapolis, MN, USA); MUC2 (Gene Tex, Irvine, CA, USA); vimentin (Sigma-Aldrich); α-smooth muscle actin (SMA) (DAKO, Glostrup, Denmark); LGR5 (Abgent, San Diego, CA, USA); CD44 (Bethyl Laboratories, Montgomery, TX, USA). Secondary antibodies were as follows: Alexa Fluor 488 goat anti-mouse IgG; Alexa Fluor 488 goat anti-rabbit IgG; Alexa Fluor 568 goat anti-rabbit IgG; Alexa Fluor 488 donkey anti-goat IgG (Invitrogen).
5
Liver Cancer Cell Line Treatment
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The human liver cancer cell lines HuH1 and HuH7 were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan) and routinely cultured with Dulbecco's modified Eagle's medium supplemented with 10% FBS. Epirubicin and 5-fluorouracil (5-FU) were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) and Kyowa Kirin (Tokyo, Japan), respectively. HuH1 and HuH7 cell lines were seeded at 10,000 cells per well in a 6-well plate treated with Epirubicin (0.5 μg/ml for HuH1 and 0.1 μg/ml for HuH7) or 5-FU (2.0 μg/ml for HuH1 and 2.5 μg/ml for HuH7) for 5 days based on IC50 data obtained from a cell proliferation assay. These cells were then used for quantitative reverse transcription-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and immunofluorescence analyses, as described previously [12] (link).
6
Murine Colon26 Tumor Models for Immunotherapy
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We established mouse tumor ascites models by subcutaneously (s.c.; 5 × 105) and intraperitoneally (i.p.; 2 × 105) implanting with Colon26 cells to observe both solid and i.p. tumors simultaneously. Treatments with agents were started on day 3–5 after tumor implantation. The following agents were used: Anti-PD1 mAb (Clone 29F.1A12; BioLegend), anti-LAG3 mAb (Clone C9B7W; BioXCell), anti-TIGIT mAb (Clone 1G9; BioXCell), mouse IgG (mIgG, Clone MOPC-21; BioXCell), an AURKA inhibitor MLN8237 (Selleck), a CDK4/6 inhibitor PD0332991 (AdooQ), and 5-fluorouracil (5-Fu; Wako). To deplete CD8+ T cells and NK cells, mice were i.p. injected with anti-CD8 mAb (Clone 2.43, 200 µg; BioXCell) or anti-asialo GM1 polyclonal Ab (20 µL; BioLegend) before and during the treatments. The depletion efficacy (> 80%) was validated by flow cytometry. Tumor size was measured (0.5 x Length x Width2, mm3), and mouse survival was observed. Two weeks after tumor implantation, s.c. tumors, spleens and peritoneal exudate cells (PECs) were harvested from the mice for assays. Splenic CD8+ T cells (2 × 105) were stimulated with the H-2Ld-restricted tumor antigen AH1 peptides (MBL), and were tested for cytotoxic activity (target = Colon26, 4 h) as described before [14] (link). Cytotoxic activity of NK cells was similarly assessed using Yac-1 cells as a target.
7
Lung Cancer Cell Line Assay Protocol
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A549 was purchased from the American Type Culture Collection (Manassas, VA). PC3, PC9, and PC14 were purchased from Immuno-Biological Laboratories (Takasaki, Japan). RERF-LC-KJ, and RERF-LC-MS were purchased from the Human Science Research Resources Bank (Osaka, Japan). A549-luc-C8 was purchased from Caliper Life Sciences (Hopkinton, MA). Anti-actinin-4 rabbit polyclonal antibody (Ab-2) and mouse monoclonal antibody (13G9, Abnova, Taiwan) were produced as described previously [12 (link), 20 (link), 21 (link)]. Anti-β-actin mouse monoclonal antibody (AC-15), anti-Cdc42 mouse monoclonal antibody (M152) and anti-cytokeratin 19 rabbit polyclonal antibody (ab53119) were purchased from Abcam (Cambridge, UK). Cisplatin (Nippon Kayaku, Japan), vinorelbin (Kyowa Hakko Kirin, Japan) and 5-fluorouracil (5FU, Wako Pure Chemical Industries, Japan) were purchased as indicated.
8
Dissolution of Chemotherapeutic Agents
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DMSO (07–4860-5; Sigma-Aldrich, St. Louis, MO) was used as a solvent of chemical compounds. Docetaxel (DTX, 047-31281), paclitaxel (PTX, 163-28163), 5-fluorouracil (5-FU, 068-01401), and cisplatin (CDDP, 033-20091; all purchased from Fujifilm Wako Pure Chemicals, Osaka, Japan) were dissolved in DMSO or saline before being used for the experiments. A potent inhibitor of FOXM1, thiostrepton (T8902; Sigma-Aldrich), was dissolved in DMSO before its use.
9
Extraction and Cytotoxicity of Kaempferia Galanga
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Ethanol (95%) used for extraction of Kaempferia galanga Linn. rhizome was obtained from Labscan Asia Co. Ltd. (Bangkok, Thailand). HPLC grade methanol and distilled water were purchased from Fisher Scientific (Leicestershire, UK). The cell culture reagents including RPMI 1640, DMEM, fetal bovine serum (FBS), phosphate buffer saline (PBS), penicillin and streptomycin antibiotics were purchased from Gibco Life Technologies (NY, USA). The cholangiocarcinoma cell line (CL-6) was kindly provided by Associate Professor Adisak Wongkajornsilp, Department of Pharmacology, Faculty of Medicine, Siriraj Hospital, Bangkok, Thailand. Methyl thiazoldiphenyl tetrazolium (MTT) was obtained from Life Technologies (CA, USA) and dimethyl sulfoxide (DMSO) was purchased from MP Biomedicals (CA, USA). 5-Fluorouracil (5-FU) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Tween-80 was purchased from Sigma-Aldrich (MO, USA). Neutral buffered formalin (NBF, 10%) was purchased from Bio-Optica (Milano, Italy). Isoflurane for euthanasia was purchased from Minrad Inc. (PA, USA). All other chemicals and reagents were high purity grade obtained from commercial suppliers. The standard ethyl-p-methoxy cinnamate (EPMC) was kindly supplied by Dr. Sumet Kongkiatpaibo, Drug Discovery and Development Center, Thammasat University, Thailand.
10
Antibody Validation and Reagent Sourcing
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Antibodies against Rab1A, ubiquitin, Hsp72, and p62 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against all forms of poly (ADP-ribose) polymerase-1 (PARP-1) and cleaved forms of caspase-3 were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against β-actin and LC3B antibody were purchased from Sigma (St. Louis, MO). Anti-Rab1B antibody was purchased from Abgen (San Diego, CA). HRP-conjugated secondary antibodies were purchased from GE Healthcare Bio-Science (Little Chalfont, Bucks, UK). Two different anti-Hsc70 antibodies used in affinity purification were produced in our laboratory [25] (link), and anti-Hsc70 antibody used in the other experiments was purchased from Enzo (Farmingdale, NY). MG132 was purchased from Sigma (St. Louis, MO). 5-Fluorouracil (5-FU), and brefeldin A (BFA) were obtained from Wako (Osaka, Japan), diluted in dimethyl sulfoxide (DMSO), and stored at −20°C.
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