Cell Culture Incubation with SCFA

10 III. MATERIALS AND METHODS

A. MATERIALS AND EQUIPMENTS

1. MATERIALS

The sample used for this research was the fermentation product of type III resistant starch RS3 derived from Ipomoea batatas var. Jago provided by Indonesian Center of Agricultural Post Harvest Research and Development, Bogor in the form of cell free supernatant consisted of 52.95 mM acetic acid, 66.93 mM propionic acid, and 92.41 mM butyric acid. HCT-116 colon cancer cells was provided by Primate Research Center, Bogor Agricultural University. Materials used in this research were Dulbecco‟s Modified Eagle‟s Medium DMEM Gipco USA, Fetal Bovine Serum FBS Gipco USA, antibiotics penicillin and streptomycin Invitrogen USA, Phosphate Buffered Saline PBS Gipco USA, Trypsin 0.25 Difco USA, Qiagen DNA extraction kit lysis buffer, Aw 1, Aw 2, and AE buffer, agarose powder Promega, ethidium bromide Gipco USA, gel loading buffer, double-distilled water, and TAE buffer 50x Gipco USA 0.484 gram Tris-Base, 0.1142 mL acetic acid, and 0.2 mL 0.5M EDTA pH 8.0.

2. EQUIPMENT

The equipments used in this research were biosafety cabinet, dissposable gloves, dissposable pipettes 1 mL, 2 mL, 5 mL, 10 mL, 25 mL, and 50 mL, micropipette, T25 and T75 tissue culture flask, incubator 37 o C, 5 CO 2 , refrigerator, water-bath, inverted microscope, centrifuge, centrifuge tube, glass bottle, gel doc, cuvette, and DNA electrophoresis.

B. METHODS

1. Cell Culture

HCT-116 colon cancer cells were warmed up from cryogenic state using waterbath with 37 ºC. Then, the cells were pelleted using centrifuge 750 g and media was discarded. HCT-116 colon cancer cells have been cultured in Dulbecco‟s Modified Eagle‟s Medium DMEM supplemented with 10 Fetal Bovine Serum FBS and 1 antibiotics penicillin 100 unitmL; streptomycin 100µgmL. Cells 1 x 10 5 were seeded into T25 tissue culture flask. The cells were incubated at 37 o C in 5 CO 2 incubator. The growth medium was changed every 2-3 days.The cells were subcultured every week or when the cells already reach 100 confluency. Subculture consisted of several steps. First, the medium were discarded and the cells were washed using Phosphate Buffered Saline PBS to make sure that there wasn‟t any media left in the flask. Next, the cells were trypsinized with Trypsin 0,25 to free the cells from adhesion to the flask followed by incubation in 37 ºC for 5-10 minutes. After that, the sample was centrifuged 750 g for 5 minutes used to pellet the cells, and the supernatant was discarded. The pellet were resuspended using fresh medium and counted using 11 hemocytometer to seed the cell into flasks in an acurate amount. 1 × 10 5 cells were seeded into T25 flask every subculture for maintaining.

2. Incubation with SCFA

Analysis was done using eight T25 flasks which contained 1 x 10 5 cells as the starting cell number. The flasks was incubated for 2 x 24 hr or to reach 50 confluency. Next, incubation was continued for another 4 x 24 hr in the absence control or presence of various concentration of cell free supernatant contained SCFA 0.625 mM, 1.00 mM, and 1.25 mM butyrate in the cell free supernatant. For the treatment of the experimental cultures, the cell free supernatant contained SCFA was diluted with DMEM. At the end of 4 x 24 hr incubation period, the cells were observed using inverted microscope with magnification 4x10. Then, the medium contained of dead cells was collected into centrifuge tube and the attached cells were washed with phosphate buffered saline PBS. The medium contained of dead cells that floating in the medium and made the medium became turbid. After that, the attached cells were trypsinized with trypsin 0.25 to free the cells from adhesion to the flask followed by incubation in 37 ºC for 5-10 minutes and pipetted into centrifuge tube. Next, the attached cells in the trypsin and the medium were pelleted with centrifugation 750 g for 5 minutes. After that, the supernatant was discarded. The pellet were resuspended using 10 mL of PBS steril and centrifuged as before. Then, the supernatant was discarded. The pellet were resuspended using 1 mL PBS steril, then pipetted into eppendorf. Next, attached and dead cells were centrifuged 1000 g for 5 menit. The supernatant was discarded. And the last, the cuvettes were stored at -20 o C.

3. DNA Extraction Qiagen DNA Extraction Kit