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  • Talanta br Materials and methods br Cell lines and

    2020-08-12

     Talanta 199 (2019) 634–642
    2. Materials and methods
    2.1. Cell lines and buffers
    The human gastric cancer cell lines BGC-823 and SGC-7901 were used for the selection. The other cell lines used in this study include the human gastric cancer cell lines MGC-803 and MKN28, the human col-orectal cancer cell lines SW620, HT29 and CCL187, the human em-bryonic kidney cell line HEK293, the Chinese hamster ovary cell line CHO, the African green monkey kidney fibroblast cell line COS-7, the murine fibroblast cell line NIH3T3, and the human normal gastric epithelial cell line GES-1. BGC-823, SGC-7901, MGC-803, MKN28, SW620, HEK293, CHO, COS-7 and NIH3T3 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HT29 and CCL187 were purchased from American Type Culture Collection (ATCC). All Pepstatin-A were cultured at 37 °C in a 5% CO2 atmosphere. The growth medium used for BGC-823, MGC-803, SGC-7901, MKN28, GES-1, NIH3T3 and HEK293 was high-glucose DMEM (GIBCO) containing 10% FBS (GIBCO) and 100 units/mL penicillin-streptomycin (GIBCO). The growth medium for the other cells was PRMI1640 (GIBCO) con-taining 10% FBS and 100 units/mL penicillin-streptomycin.
    Washing buffer (WB) was prepared from Dulbecco's phosphate buffered saline (PBS) with additional glucose (4.5 g/L) and MgCl2 (5 mM). Binding buffer (BB) was prepared by adding salmon sperm DNA (0.1 mg/mL) and BSA (1 mg/mL) to the washing buffer.
    2.2. Tissue microarray
    The tissue microarray (TMA) was purchased from Shanghai Biotech Company, Ltd. (Shanghai, China). The TMA contains fifteen cases of GC specimens and corresponding adjacent tissues (HStmA030PG03), of which eight cases were well/moderately differentiated and seven cases were poorly differentiated.
    2.3. Random DNA library and primers
    An initial ssDNA library (5′-AAGGAGCAGCGTGGAGGATA-45N-TTAGGGTG TGTCGTCGTGGT-3′) consisting of 45-base randomized sequences was synthesized by Sangon Biotechnology Co., Ltd. (Shanghai,China), where N represents a randomized nucleotide of either A, G, C, or T. The forward primer (5′-AAGGAGCAGCGTGGAGG ATA-3′) and the biotin-labelled reverse primer (5′-Bio-ACCACGACGA CACACCCTAA-3′) were used for PCR amplification of the DNA library or to separate the single-stranded DNA by streptavidin-coated se-pharose beads (GE Healthcare, USA). The FAM-labelled forward primer was used to monitor the progress of selection by flow cytometry (BD, USA).
    2.4. Subtractive Cell-SELEX procedures
    The subtractive Cell-SELEX procedure was performed as previously described [19]. Briefly, an initial ssDNA library (12 nmol) was first dissolved in 1 mL of pre-cooled BB. Then, the DNA was denatured by heating at 95 °C for 5 min and subsequently cooled on ice for 10 min. The library was incubated with BGC-823 cells at 4 °C for 1 h, and then the cells were washed with WB to remove the unbound ssDNA se-quences. Adherent BGC-823 cells were scraped off and re-suspended in 500 µL of DNase-free deionized water. The cell suspension was then heated at 95 °C for 10 min and centrifuged at 12,000 rpm for 5 min. The supernatant, which contained the eluted ssDNA aptamers, was collected and then amplified by PCR using biotin- and FAM-labelled primers. After isolation using streptavidin-coated sepharose beads, the FAM-la-belled ssDNA pool was desalted with an NAP-5 column (GE Healthcare, USA) and collected for the next round of selection.
    From the fourth round of selection, the selected ssDNA pool was first incubated with subtractive SGC-7901 cells to perform subtractive
    selection, thereby filtering out ssDNA sequences that may bind to the subtractive SGC-7901 cells. The unbound ssDNA pool was specifically targeted to BGC-823 cells for positive selection. Pepstatin-A Furthermore, the target cell number and the concentration of the ssDNA pool were gradually reduced with the selective round proceeding 15 cycles.
    To select aptamers that sensitively and specifically recognize BGC-823 cells, the selection pressure was progressively enhanced by 1) de-creasing the concentration of targeted BGC-823 cells from 107 to 105, 2) decreasing the amount of the ssDNA pool from 1200 pmol to 50 pmol,
    3) decreasing the incubation time with the target BGC-823 cells from 1 h to 20 min, 4) increasing the amount of BSA and salmon sperm DNA in the incubation buffer, and 5) increasing the number of washes after the incubation from three to six (Table S1).
    2.5. Flow cytometry analysis
    To monitor the binding of the enriched ssDNA pool during Cell-SELEX, FAM-labelled ssDNA pools of 3, 6, 9, 12, and 15 rounds were incubated with the target BGC-823 cells or the subtractive SGC-7901 cells in 200 µL of BB at 4 °C for 30 min. Then, the cells were washed twice after incubation and analysed by flow cytometry. The FAM-la-belled ssDNA library was used as a negative control. All experiments were repeated three times.