Anticancer effect of ALF, Se NPs and ALF-Se NPs was assayed using three human cancer cell lines, including breast cancer cell line (MCF-7), liver cancer cell line (HepG-2) and colon cancer cells (Caco-2). All cell lines were obtained from the American Type Culture Collection (ATCC, USA). MCF7 and HepG2 were maintained in RPMI (Lonza, USA) while Caco2 cell line was cultured in DMEM (Lonza, USA), both media were supplemented with 10% FBS. All cancer cells (5 × 103 cells/well) were seeded in sterile 96-well plates. After 24 h, serial concentrations of ALF (62.5, 125, 150, 500, 1000 and 2000 ug/ml), Se NPs and ALF-Se NPs (31.25, 62.5, 125, 150, 500 and 1000 ug/ml) as compared to 5-FU (standard reference drug) were incubated with four cancer cell lines for 72 h at 37 °C in 5% CO2 incubator. MTT method was done as described above. The half maximal inhibitory concentration (IC50) values were calculated using the Graphpad Instat software. Furthermore, cellular morphological changes before and after treatment with ALF, Se NPs and ALF-Se NPs were investigated using phase contrast inverted microscope with a digital camera (Olympus, Japan). Importantly, selectivity index (SI) that defined as the ratio of the IC50 on normal cells versus tumor cells of ALF with or without Se NPs was estimated as IC50-N/IC5075 (link).
Caco 2
Manufactured by Lonza
Sourced in Belgium, United States
Caco-2 is a cell line derived from human colorectal adenocarcinoma. It is widely used in the pharmaceutical industry as an in vitro model for assessing intestinal absorption and permeability of drug candidates.
Automatically generated - may contain errors
Lab products found in correlation
Caco 2, by American Type Culture Collection (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
L glutamine, by Lonza (4 mentions)
Ht 29, by Lonza (4 mentions)
Hepg2, by Lonza (3 mentions)
Penicillin, by Lonza (3 mentions)
Streptomycin, by Lonza (3 mentions)
Penicillin streptomycin, by Lonza (3 mentions)
Fetal bovine serum (fbs), by Lonza (3 mentions)
Foetal bovine serum (fbs), by Lonza (2 mentions)
Sw620, by Lonza (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Sw480, by Lonza (2 mentions)
Non essential amino acids (neaa), by Lonza (2 mentions)
Mccoy medium, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Be12 611f 12, by Lonza (2 mentions)
Ht 29, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Lonza (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
16 protocols using caco 2
1
Anti-cancer Potential of ALF, Se NPs, and ALF-Se NPs
2
Glucose Dosage Effects on Colon Cancer Cells
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The American Type Culture Collection provided the colon cancer cell line Caco2, SW620, and normal colon cells FHC (ATCC, Manassas, VA, USA). The cells were grown in DMEM media with low glucose content (1.0 g/L, 5.6 mmol/L). In addition, 10 percent foetal bovine serum (FBS) (Lonza Bioproducts), penicillin 100 U/mL, and streptomycin 100 mg/mL (Lonza Bioproducts) were added to the media, which was incubated at 37 °C in a humidified chamber with 5% CO 2 .
Caco2, SW620, and FHC colon cancer cell lines were planted in 6-well plates as indicated above. All cell lines were cultured for 24 hours (3 wells per treatment) with different doses of D-(+)-glucose [5 Mm (90 mg/dL), 10 mM (181 mg/dL), and 15 mM (271 mg/dL)].
Caco2, SW620, and FHC colon cancer cell lines were planted in 6-well plates as indicated above. All cell lines were cultured for 24 hours (3 wells per treatment) with different doses of D-(+)-glucose [5 Mm (90 mg/dL), 10 mM (181 mg/dL), and 15 mM (271 mg/dL)].
3
Culturing Human Colon Cancer Cell Lines
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Eight human colon cancer cell lines, SW1116, SW480, DLD-1, SW620, HT-29, Caco-2, COLO205 and T84 were obtained from the American Type Culture Collection (ATCC). SW1116, SW480, DLD-1, SW620, HT-29 and Caco-2 were cultured in DMEM media (Lonza) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% L-glutamine (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich). COLO205 was maintained in RPMI 1640 media (Lonza) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin/streptomycin. T84 was maintained in DMEM/Ham's F12 media (Lonza) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin. Cells were grown at 37°C in a humidified atmosphere of 5% CO2.
Dukes' stage and tissue origin for each cell line is shown inTable S1 .
Dukes' stage and tissue origin for each cell line is shown in
4
Evaluating Cytotoxicity of Coaxial NFs
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To investigate the potential biological application of the coaxial GDD NFs, in vitro cytotoxicity study was done for GDD, the plain and medicated coaxial NFs using methyl thiazolyl tetrazolium (MTT) assay. A Human colorectal adenocarcinoma (Caco-2) was obtained from the American Type Culture Collection (Manassas, VA, USA). Caco-2 cells were cultured in complete cell culture medium (DMEM+) composed of: Dulbecco Modified Eagle’s Minimal Essential medium (DMEM; 4.5 g/L glucose) supplemented with L-glutamine (Lonza, Belgium), non-essential amino acids (NEAA; 1% v/v; BioWhittaker, Lonza), penicillin-streptomycin (PEST; 1% v/v; Sigma, MO, USA), and heat-inactivated fetal bovine serum (FBS; 10% v/v; Gibco, MA, USA). Culture medium was refreshed every two day (at 80–85% confluence) using 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA).
5
Coculture of Colon Cell Lines and Primary Mast Cells
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Human colon cancer cell lines HT29 and Caco2, and human normal colon cell line CCD841 were obtained from American Type Tissue Culture Collection. HT29 were grown in McCoy medium (Gibco) supplemented with heat-inactivated 10% fetal calf serum (FCS), and 100 μg/ml pen/strep (penicillin and streptomycin, Gibco, 26600080). Caco2 and CCD841 were cultured in EMEM medium (Lonza, BE12-611F/12) supplemented with 10% FCS, 100 μg/ml pen/strep, 1% non-essential amino acids, 1% L-glutamine and 1% sodium pyruvate (Gibco, 11360-039). All cells were cultured in a humidified 37°C/5% CO2 incubator. Prior to coculture 1 × 105 human colon cells were seeded in a 24-well plate. Standard culture medium were removed after 24 h and cells were washed with PBS. Thereafter 1 × 106 primary human MC resuspended in SFEM supplemented with SCF were added to the colon monolayer. Cells were harvested after 72 h of coculture and stained with an apoptotic cell dye YO-PRO1 and antibodies of FcεRIa and CD117. The subset of CD117+ FcεRIa+ cells was gated by flow cytometry and analyzed for expression of Siglec-6 (R&D, FAB2859P).
6
Culturing Caco-2 Colorectal Cancer Cells
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Human colorectal adenocarcinoma cells (Caco-2, American Type Culture Collection (ATCC), HTB-37) were cultured at 37 °C in a humidified atmosphere with 5% CO2 in a complete medium containing Dulbecco’s Modified Eagle Medium (DMEM, high glucose, GlutaMax, pyruvate; Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS, Thermo Fisher Scientific), 1% (v/v) penicillin-streptomycin (Thermo Fisher Scientific) and 15 mM HEPES (Thermo Fisher Scientific). Caco-2 cells were used in the experiments from passages 50 to 60, and were regularly tested for mycoplasma using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza).
7
Comprehensive Cell Line Validation Protocol
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All the cell lines used in this study were obtained from Holding Company for Biological Products and Vaccines VACSERA, Egypt; MCF-7 cells (Human breast adenocarcinoma), HepG2 (human hepatocellular carcinoma), Caco-2 (colon carcinoma), A549 (human lung adenocarcinoma), PANC-1 (human pancreatic cancer), Vero cells (derived from normal kidney cells).
The RPMI 1640 medium (Lonza, Switzerland) was used to maintain all cell lines. The media also contained 10% fetal bovine serum (Gibco, USA), 1% penicillin, and 1% streptomycin (Sigma Aldrich, USA). In a humidified cell incubator with a 5% Carbon dioxide environment, the cells were kept at 37 °C. The study was examined and approved by the Ethics committee in the Faculty of Pharmacy-October University for Modern Sciences and Arts (MSA) with ethics approval number (BP2/Ec2/2021PD).
The RPMI 1640 medium (Lonza, Switzerland) was used to maintain all cell lines. The media also contained 10% fetal bovine serum (Gibco, USA), 1% penicillin, and 1% streptomycin (Sigma Aldrich, USA). In a humidified cell incubator with a 5% Carbon dioxide environment, the cells were kept at 37 °C. The study was examined and approved by the Ethics committee in the Faculty of Pharmacy-October University for Modern Sciences and Arts (MSA) with ethics approval number (BP2/Ec2/2021PD).
8
Culturing Human Colon Cancer Cell Lines
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Human colon cancer cell lines, HT29 and Caco2, were obtained from American Type Tissue Culture Collection. HT29 were grown in McCoy medium (Gibco) supplemented with heat-inactivated 10% fetal calf serum (FCS), and 100 μg/ml pen/strep (penicillin and streptomycin, Gibco, 26600080). Caco2 were cultured in EMEM medium (Lonza, BE12-611F/12) supplemented with 10% FCS, 100 μg/ml pen/strep, 1% non-essential amino acids, 1% L-glutamine and 1% sodium pyruvate (Gibco, 11360–039). All cells were cultured in a humidified 37°C/5% CO2 incubator. Green fluorescent protein (GFP) expressing HT29 were generated by transfection with pMONO-neo-GFP plasmid containing GFP and neomycin resistance genes (Invivogen, pmonon-gfp) by electroporation (Nucleofector, Lonza, Belgium). GFP-transfected HT29 were then selected under Geneticin (Gibco, 10131035).
9
Cell Culture and Differentiation Protocol
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HepG2 (hepatic) and Caco‐2 (intestinal) cells were purchased from American Type Culture Collection (ATCC; USA). Cells were maintained in Dulbecco's modified eagle's medium (DMEM; Sigma; Dorest, UK) supplemented with 10% foetal bovine serum (FBS; Bio‐Whittaker, Berkshire, UK) for HepG2 cells, and 15% sterile filtered FBS for Caco‐2 cells. THP1 (monocyte) cells were purchased from the European Collection of Cell Culture (ECACC; Porton Down, UK) and grown in RPMI‐1640 (Sigma; Dorest, UK) supplemented with 10% sterile filtered FBS. Adherent cells (HepG2 and Caco‐2) were routinely sub‐cultured every 4 days when 95% confluent. THP1 cells were sub‐cultured when a density of 1 × 106 cells/mL was achieved. THP1 cells were activated to macrophage‐like cells (ATHP1) by the addition of phorbol 12‐myristate 13 acetate (PMA: Sigma; Dorest, UK) to a final concentration of 10nM in THP1 culture medium (RPMI‐1640 supplemented with 10% sterile filtered FBS). Cells were then incubated at 37°C and 5% CO2 for 7 days prior to use to allow differentiation from monocytes to macrophage‐like cells. Cell count and viability were determined by Trypan Blue exclusion assay. The range of passage number after receipt from ATCC or ECACC was equal to 4 – 7 for HepG2, 5 – 8 for Caco‐2, 8 – 10 for THP‐1, 8 – 10 for CEM, and 8 – 10 for ATHP‐1.
10
Fluorescence Imaging of Cell Lines
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Human (Caco-2, MCF-7, and KMST-6) and Chinese Hamster Ovary (CHO) cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Caco-2 (colorectal adenocarcinoma), MCF-7 (breast carcinoma), and KMST-6 (skin fibroblast) cells were cultured in Dulbecco’s Modified Eagle’s Medium (Lonza, Verviers, Belgium), while CHO cells were cultured in Ham-F-12 media (Lonza) supplemented with 10% fetal bovine serum (Biochrom, Cambourne, UK) and 0.5% penicillin/streptomycin (Lonza). The cells were seeded on cover slips (5 mm diameter) at a cell density of 2×105 cells/mL in a six-well plate and cultured for 24 h at 37°C in a humidified incubator. The cells were fixed with 4% paraformaldehyde for 10 min and then rinsed with PBS. The cells were subsequently incubated with either AHP-QD625 (50 nM) or QD625 (50 nM), prepared in their respective media for 1 h at 37°C. The coverslips were then mounted on microscope slides using Fluoroshield™ (Sigma-Aldrich Co., St Louis, MO, USA) with diamidino-2-phenylindole (DAPI). The slides were viewed under a Zeiss Axioplan 2 fluorescence microscope at 100× magnification.
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