Accumax

Samples were stained with aggresome and aggregation of unfolded protein was evaluated by fluorescence microscope and flow cytometry according to the manufacturer’s protocol. For fluorescence microscope analysis, samples were incubated with aggresome reagent (1:2000; Enzo Life Science Inc., Farmingdale, NY, USA) for 60 minutes and slides were examined with a fluorescence microscope (DM 2500). For flow cytometry, cells were washed twice with phosphate buffered saline (PBS) and incubated with Accumax^TM (Innovative Cell Technologies, San Diego, CA, USA) for 10 minutes at 37 °C. Cells were recovered in fluorescence activated cell sorting (FACS) buffer composed of DMEM without Phenol Red (Nacalai Tesque) + 2%FBS, passed through a BD Falcon™ 70 μm cell strainer (BD Biosciences, Franklin Lakes, New Jersey, USA), and resuspended in FACS buffer. The cells were then incubated with aggresome reagent (1:2000) for 60 minutes at room, temperature, and washed three times with PBS. The cells were resuspended in FACS buffer and analyzed by flow cytometry using CellQuest Pro software (BD Biosciences).

Four-week-old male athymic NOD/SCID mice were used to establish chemotherapy-resistance model. The mice were subcutaneously injected with SW620 cells (2×10⁶ cells in 200 μL volume). After 14 days of inoculation, 5-FU by IP injection at 30mg/kg/mouse was applied thrice a week for four weeks. After four weeks, the mice were sacrificed and tumors were collected and digested into primary cells, SW620R. To obtain 5-Fu resistant cell line, tumor cells were isolated and purified by using ACCUMAX^TM (Innovative Cell Technologies) according to manufacturer's instructions. In brief, the tumor tissues were rinsed with sterile DPBS twice, and transferred to a petri dish containing sterile DPBS. The tissues were cut by surgical scissors into small pieces approximately 1 mm in size, then transferred to 50 ml sterile centrifuge tube. The pieces were settled by centrifugation and carefully removed the supernatant for two times. The pieces were transferred to new 50 ml sterile centrifuge tube and treated with ACCUMAX^TM, and then incubated on an agitator at RT for 30 minutes. After the incubation, the cells were isolated by cell strainers, then removed the supernatant by centrifugation at 900 rpm for 5 minutes. The cells were resuspended in DPBS and centrifuged for washing two times. After washing, the primary cells (SW620R) were cultured in RPMI1640 medium containing 5-FU (concentration 1 μM).

Cell apoptosis was evaluated with Annexin V (Medical & Biological Laboratories Co., Ltd.) staining. For fluorescence microscopy analysis, the cells were incubated with DMEM supplemented with Annexin V for 30 minutes, followed by fixation with 4% paraformaldehyde for 10 minutes. The slides were examined with a fluorescence microscope (DM 2500; Leica Microsystems). For flow cytometry, cells were incubated with DMEM supplemented with Annexin V for 15 minutes, and harvested by digestion with Accumax^TM (Innovative Cell Technologies). Recovered cells were then analyzed by flow cytometry using CellQuest Pro software (BD Biosciences).