Methods and applications for in situ labeling of cells
An in situ labeling and cell technology, applied in the biological field, can solve the problems of limitation and cytotoxicity, and achieve high-sensitivity and high-resolution positioning and tracking, low cytotoxicity, and clear labeling effect
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Embodiment 1
[0060] A kind of synthetic of choline analogue (AE-Cho), comprises the steps:
[0061] 1) Take 8.5mL (98.52mmol) of 1,2-dibromoethane and 3.2g (49.22mmol) of sodium azide into 25mL of dimethylformamide (DMF) organic solvent, stir at 80°C for 20h, Then cooled to room temperature; then extracted three times with sodium chloride and ice pentane, the organic layer was washed three times with aqueous sodium chloride, dried over sodium sulfate and concentrated under reduced pressure to obtain purified 1-azido-2-bromoethyl Alkane (yield 81.21%);
[0062] 2) Add 49.22 mmol of dimethylethanolamine to 15 mL of tetrahydrofuran (THF), then add 1-azido-2-bromoethane purified in step (1), and stir at 0°C for 6 hours under argon atmosphere; the reaction is over Obtain white precipitation product afterward, wash three times with ether, vacuum-dry, obtain the choline (AE-Cho) solid (yield 70.5%) of pure azidoethyl modification, the structure of described AE-Cho is shown in P2 :
[0063] ...
Embodiment 2
[0068] A kind of synthetic of choline analogue (AP-Cho), comprises the steps:
[0069] 1) Add 98.52mmol of 1,2-dibromopropane and 49.22mmol of sodium azide into 25mL of dimethylformamide (DMF) organic solvent, stir at 80°C for 20h, then cool to room temperature; Sodium and ice pentane were extracted three times, the organic layer was washed three times with sodium chloride aqueous solution, dried over sodium sulfate and concentrated under reduced pressure to obtain purified 1-azido-2-bromoethane (yield 75.59%);
[0070] 2) Add 49.22 mmol of dimethylethanolamine to 15 mL of tetrahydrofuran (THF), then add the 1-azido-2-bromopropane purified in step (1), and stir the reaction at 0°C for 6 h under argon atmosphere; after the reaction A white precipitated product was obtained, washed three times with ether, and dried in vacuo to obtain a pure azidopropyl-modified choline (AP-Cho) solid (69.32% yield). The structure of the AP-Cho is shown in P3:
[0071]
[0072] The synthetic ...
Embodiment 3
[0076] A method for in situ labeling of human breast cancer cells (MCF-7), comprising the steps of:
[0077] 1) MCF-7: The culture medium is DMEM medium containing 10% calf serum, culture conditions: 37°C, 5% CO 2 / 95% air, saturated humidity, replace the culture medium every other day, and subculture once every 3 to 4 days;
[0078] 2) Metabolic binding of choline analogs in MCF-7 cells: first wash the cells 3 times with PBS buffer, and replace with fresh DMEM medium without calf serum;
[0079] Set up an experimental group and a control group, wherein, the experimental group adds the AE-Cho prepared in Example 1 to the cell culture medium, and the control group uses DMEM medium without calf serum instead of the AE-Cho prepared in Example 1 to add the cell culture medium ;
[0080] Co-cultivate for 24 h, then wash off excess AE-Cho with PBS buffer (wash the cells 2 to 4 times with PBS buffer), add fresh medium to the final cell concentration of 1×10 6 ,spare;
[0081] 3) ...
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