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).
Irinotecan
Manufactured by LC Laboratories
Sourced in Japan
Irinotecan is a chemical compound used in laboratory research and analysis. It is a topoisomerase I inhibitor, which means it disrupts the function of the enzyme topoisomerase I, an essential enzyme involved in DNA replication and transcription. Irinotecan is commonly used in cell culture and biochemical assays to study the effects of topoisomerase I inhibition on various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Primocin, by Thermo Fisher Scientific (2 mentions)
Nicotinamide, by Merck Group (2 mentions)
Sb202190, by Cayman Chemical (2 mentions)
A83 01, by Adooq (2 mentions)
Y 27632, by Cayman Chemical (2 mentions)
Vinblastine, by Merck Group (2 mentions)
Mitoxantrone, by Selleck Chemicals (2 mentions)
Nocodazole 74151, by Merck Group (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
α smooth muscle actin, by Agilent Technologies (1 mentions)
Vimentin, by Merck Group (1 mentions)
Mouse egf, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
E cadherin, by R&D Systems (1 mentions)
5 fluorouracil 5 fu, by Fujifilm (1 mentions)
Ifn γ, by Merck Group (1 mentions)
5 fluorouracil 5 fu, by Merck Group (1 mentions)
Oxaliplatin, by Merck Group (1 mentions)
6 protocols using irinotecan
1
Organoid Generation from Colorectal Tissues
2
Drug Concentration Optimization for Cell Studies
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Stock concentrations of the compounds were prepared in Dimethyl sulfoxide (DMSO) and stored at −20°C. SM-7368 (481411-5mg), CPT (C9911-250MG), KU60019 (SML1416-5MG), irinotecan (IRI) (SKU-I1406-50MG), and VE-822 (1232416-25-9) were purchased from Sigma-Aldrich (St. Louis, MO). AZD7762 (Enzo, ENZ-CHM185-0005) from ENZO. Flow Cytometry Staining Buffer (1X) (Cat# FC001), Mouse PE-IgG2a (R&D IC003p), and Human HLA Class I (MHC I) Phycoerythrin mAb (Clone W6/32) were purchased from R&D Systems. Unless indicated otherwise, the drugs were used at the following concentrations: CPT 0.5-1 µM; SM-7368 5 µM; irinotecan 25 µg/ml; VP-16 (Etoposide, LC laboratories) 25 µM; γ-IFN (Sigma) 50 ng/ml; VE822 (Selleckchem) 100 nM; AZD7762 100 nM, KU60019 at 1 µM; erlotinib (LC laboratories) 20 µM; oxaliplatin (Sigma) 10 µM; taxol (Sigma) 50 nM; 5-Fluorouracil (5-FU, Sigma) 10 µM. For experiments in this study, single drug concentrations were pre-determined based on the length of treatment (24–48 h) that induced the expression of target proteins without visibly killing the treated cells.
3
Colorectal Tumor-Derived Organoid Culture
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Human colorectal tumor tissue-derived ALI organoids were cultured as described previously [10 (link)]. The medium 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 epidermal growth factor (EGF) (PeproTech, Inc., Rocky Hill, NJ, USA); 500 nM A83-01 (Adooq Bioscience, Irvine, CA, USA); and 3 μM SB202190; 10 μM Y-27632 (Cayman, Ann Arbor, MI, USA). Stem cell-related signal inhibitors were as follows: YO-01027 (Toronto Research Chemicals, Toronto, Canada); DAPT (Adooq Bioscience); WAV939; Wnt-C59; AY9944; and GANT61 (Cayman). Anti-cancer drugs were as follows: 5-FU (WAKO, Tokyo, Japan); Irinotecan (LC Laboratories, Woburn, MA, USA); and Oxaliplatin (Adooq Bioscience). Antibody sources were as follows: GLI-1 (Gene Tex, Irvine, CA, USA); CD44 (Bethyl Laboratories, Montgomery, TX, USA); c-Myc; and Nanog (Cell Signaling, Beverly, MA, USA). Secondary antibodies were as follows: Horseradish peroxidase (HRP) conjugated anti-rabbit IgG; HRP conjugated anti-goat IgG (Cayman); and HRP conjugated anti-mouse IgG (Millipore, Temecula, CA, USA).
4
Cytotoxicity Assay of HiPco SWCNT
5
Mitotic Inhibitor Screening Protocol
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Ispinesib (S1452) and mitoxantrone (S2485) were purchased from Selleck Chemicals. Nocodazole (74151), paclitaxel (T7402), and vinblastine (V-1377) were purchased from Sigma. Irinotecan was purchased from LC Laboratories (I-4122). All compounds were dissolved in dimethylsulphoxide (DMSO).
6
Mitotic Inhibitor Screening Protocol
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