br Subtractive Cell SELEX procedures br
2.4. Subtractive Cell-SELEX procedures
The subtractive Cell-SELEX procedure was performed as previously described . 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 Palmitic acid 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. 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.
The FAM-labelled selected aptamers were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). To determine the Kd value of the selected aptamers, BGC-823 cells were incubated with various concentrations of FAM-labelled aptamers in BB at 4 °C for 30 min, and the fluorescence intensity was then determined by flow cytometry. The ssDNA library was used as the negative control. Then, the equilibrium dissociation constant (Kd) of each aptamer was determined by nonlinear regression of one-site binding, according to the following equation, using Sigma Plot 10.0 software (Jandel Scientific, UK): Y = Bmax × X/ (Kd + X), where Bmax is the saturated binding, Y is the fluorescence intensity and X is the concentration of aptamers. All experiments were repeated three times. To determine the cell specificity of the selected aptamers, cancer cell lines, including MGC-803, MKN28, SW620, HT29, CCL187, and normal cell lines, including GES-1, CHO, COS-7, HEK293, and NIH3T3, were used to analyse the binding ability with flow cytometry, as de-scribed above. All experiments were repeated three times.
2.6. Fluorescence microscopy analysis
The cells were seeded in the chamber and cultured overnight. After being washed twice with WB, the cells were incubated with 250 nM FAM-labelled aptamers or ssDNA library at 4 °C for 1 h. Then, after being washed twice with WB, the cells were fixed with 4% for-maldehyde for 15 min and stained with DAPI for 5 min to counterstain the nucleus. The fluorescence images of the cells were obtained with an Olympus IX51 fluorescence microscope (Olympus, Japan).