Anthrone approach as described by Enneb, et al. [15]. The total carbohydrates content material in

Anthrone approach as described by Enneb, et al. [15]. The total carbohydrates content material in the extracts was calculated utilizing a calibration curve of glucose and IP Agonist Synonyms expressed as percentage of carbohydrates in the extracts. The total protein content material was determined in the extracts was obtained by Bradford colorimetric assay, as described by Enneb, Drine, Bagues, Triki, Boussora, Guasmi, Nagaz and Ferchichi [15]. The total protein content material was calculated employing a calibration curve of bovine serum albumin and expressed as percentage of protein in the extracts. As for the fat content, it was determined employing Bligh and Dyer HDAC7 Inhibitor medchemexpress method for total lipids analysis as described by Chua, et al. [16]. The total fat content was determined gravimetrically and expressed as percentage of fat within the extracts. Additionally, the moisture and ash quantification was also performed as described by Ferreira, et al. [17], determined gravimetrically and expressed as percentage within the extracts. Lastly, the power value of every single extract was determined as described by Demoliner, et al. [18] utilizing the Equation (2): Total energy (kcal/100 g) = 4 ( g carbohydrates + g proteins) + 9 ( g Fat) 2.6. Cell Culture, Cell Therapy and Determination of Citotoxicity Human Caco-2 cell line (TC7 clone) was kindly supplied by Dr. Edith Brot-Laroche (UniversitPierre et Marie Curie-Paris six, UMR S 872, Les Cordeliers, France). The cells were maintained inside a humidified atmosphere of 5 CO2 at 37 C. Cells had been grown in Dulbecco’s Modified Eagles medium (DMEM) (Gibco Invitrogen, Paisley, UK) supplemented with 20 fetal bovine serum (FBS), 1 non-essential amino acids, 1 penicillin (1000 U/mL), 1 streptomycin (1000 /mL) and 1 amphotericin (250 U/mL). The cells were passaged enzymatically with 0.25 trypsin-1 mM EDTA and sub-cultured on 25 cm2 plastic flasks at a density of five 105 cells/cm2 . Culture medium was replaced each and every two days. Extract (2)Biomolecules 2021, 11,5 oftreatments had been added 24 h post-seeding for assays on Caco-2 undifferentiated cells (Garcia-Moreno et al., 2013); and 105 days post-seeding on differentiated Caco-2. Cell confluence (80 ) was confirmed by optical microscopy. The decision of those cells was related to the possibility of working with the same cell line in undifferentiated cancer cells and normal enterocytes. Extracts from avocado residues (peel, seed coat and seed) have been diluted in cell culture medium to the final concentration 1.2 mg/mL. A selection of concentrations of the avocado extracts (62.five, 125, 250, 500, and 1000 /mL) was tested. These concentrations were chosen based on preceding function, carried out by our research group, employing diverse plant extracts [191]. For cytotoxicity screening assays, the cells were seeded in 96-well plates at a density of 4 103 cells/well. Culture medium was replaced with medium containing all distinct avocado extracts and cells were incubated for 24, 48 or 72 h. Antiproliferative effect was measured using the fluorometric cell viability Resazurin assay [22]. Resazurin stock remedy at a concentration of 10 mg/mL was prepared dissolving Resazurin powder in phosphate-buffered saline (PBS) and this stock option was re-diluted 1:ten (with respect of total volume) in DMEM to acquire the operating option. Right after removing culture medium from plates, one hundred of operating option were added to each and every well and cells were incubated at 37 C for 2 h. Lastly, fluorescence was measured with excitation/emission at 544/590 nm utilizing a FLUOstar Omega (BMG Labtech).