Single-cell Glycosyl Metabolism Labeling Method Based on Glass Nanoelectrode
A nano-electrode and metabolism technology, applied in the field of live cell imaging analysis, can solve the problems of poor selectivity, long time, and inability to realize single-cell research and analysis, and achieve the effect of improving specificity and sensitivity and shortening time
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Embodiment 1
[0027] (1) According to the existing literature, draw a glass tube with a thickness of about 100 nm. A P-2000 laser puller will be used to prepare nanotubes by a single pull cycle method using quartz capillary (outer diameter 1.00mm, inner diameter 0.7mm) as raw material. The optimal pore diameter is 100nm, and the drawing parameters are Line1: heat-650, fil-3, vel-35, del-145 and pul-75; Line2: heat-920, fil-2, vel-15, del-128 and pul -200. Under this parameter, the average pore size of the nanotubes is about 100 nm.
[0028] (2) Cells were cultured by traditional methods. MCF-7 cell culture: RPMI 1640, fetal bovine serum (10%), streptomycin (100 μg mL -1 ) and penicillin (100 μg mL -1 ) to prepare culture medium. HeLa, RAW264.7 cell culture: DMEM, fetal bovine serum (10%), streptomycin (100 μg mL -1 ) and penicillin (100 μg mL -1 ) to prepare culture medium. Cells in culture flasks and culture dishes were cultured at 37°C in 5% carbon dioxide environment.
[0029] (...
Embodiment 2
[0033] (1) According to the existing literature, a glass tube of about 80 nm is drawn.
[0034] (2) Cells were cultured by traditional methods. MCF-7 cell culture: RPMI 1640, fetal bovine serum (10%), streptomycin (100 μg mL -1 ) and penicillin (100 μg mL -1 ) to prepare culture medium. HeLa, RAW264.7 cell culture: DMEM, fetal bovine serum (10%), streptomycin (100 μg mL -1 ) and penicillin (100 μg mL -1 ) to prepare culture medium. Cells in culture flasks and culture dishes were cultured at 37°C in 5% carbon dioxide environment.
[0035] (3) Centrifuge the alkyne-containing fucose (3000 rpm, 3 min) to the bottom of the tube, add 0.2 mmol of PBS buffer solution and dilute to 10 mmol. Then use a microsyringe to slowly inject 10 μL of alkyne-containing fucose from the rear end of the drawn glass tube, and then use centrifugation (3000 rpm, 3 min) to ensure that the solution can completely reach the tip of the tube. During the centrifugation process, the nanotubes were fixe...
Embodiment 3
[0039] (1) According to the existing literature, draw a glass tube with a thickness of about 100 nm.
[0040] (2) Cells were cultured by traditional methods. MCF-7 cell culture: RPMI 1640, fetal bovine serum (10%), streptomycin (100 μg mL-1) and penicillin (100 μg mL-1) to prepare culture medium. HeLa, RAW264.7 cell culture: DMEM, fetal bovine serum (10%), streptomycin (100 μg mL-1) and penicillin (100 μg mL-1) to prepare culture medium. Cells in culture flasks and culture dishes were cultured at 37°C in 5% carbon dioxide environment.
[0041] (3) After centrifuging (3000 rpm, 3 min) the azide-containing mannose (ManNAz) to the bottom of the tube, add 0.2 mmol of PBS buffer solution and dilute to 10 mmol. After slowly injecting 10 μL of ManNAz from the rear end of the drawn glass tube with a microsyringe, centrifugation (3000 rpm, 3 min) was used to ensure that the solution could completely reach the tip of the tube. During the centrifugation process, the nanotubes were fix...
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