Method and Application of Coptisine as Fluorescent Probe for Labeling Eukaryotic Cells
A technology of fluorescent probes and coptisine, applied in the field of biomedicine, can solve the problems of short storage time of reagents, inability to label live cells well, poor stability, etc., and achieve short time-consuming, easy preparation, test repeatability, and operation simple effect
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Embodiment 1
[0051] Fluorescence Spectrum of Coptisine
[0052] The coptisine structure is as follows:
[0053] Coptisine was dissolved in DMSO (Sigma), and the concentration of the mother solution was 20 mM / L. Take 2 μL of the stock solution into 2 ml DMSO so that the final concentration of coptisine is 20 μM / L. Take a new 96-well plate, and add 200 μL of diluted coptisine to each well; DMSO is used as a blank solvent control. Three replicate wells were set up in each group, and the multifunctional microplate reader (Thermo) was tested after adding the samples. First fix the wavelength of the emission light at 550 nm, and scan to obtain the excitation spectrum of coptisine; then fix the wavelength of the excitation light at 460 nm, and scan to obtain the emission spectrum of coptisine, such as Figure 1-2 . Depend on Figure 1-2 It can be seen that the excitation spectrum of coptisine has two peaks, the maximum excitation wavelength of green fluorescence is 460 nm, and the maximum em...
Embodiment 2
[0055] Spread the Hela cells into small dishes (35mm), and set up three duplicate holes for each group. Take out the cells from the incubator the next day, discard the medium, and wash once with PBS. Then use the Coptisine mother solution obtained in Example 1 to dilute to 20 μM / L with medium (DMEM (Gibco) (containing 10% FBS)), and add 1.5 ml of Coptisine diluted in the medium to each dish (dosing in reverse order, such as Add the group incubated for 8 hours first, add the group incubated for 6 hours every 2 hours, and so on), and place the cell incubator (37°C, 5% CO 2 ) for the corresponding time, and the control was to add the same volume of coptisine in the experimental group and incubate for 8 h in DMSO. Afterwards, the cells were taken out from the incubator, the medium was discarded, and washed twice with PBS. Add 500 μL 0.25% trypsin (containing EDTA) to the dish to digest for 2-5 min. Then, 500 μL of DMEM (containing 10% FBS) was added to the dish to stop the dige...
Embodiment 3
[0057] Fluorescence intensity of cells labeled with coptisine at different concentrations
[0058] Hela cells were plated into 6-well plates, and each group had three replicate wells. Take out the cells from the incubator the next day, discard the medium, and wash once with PBS. Afterwards, the mother liquor of coptisine was diluted to 20 μM / L, 10 μM / L, 5 μM / L, and 1 μM / L with culture medium (DMEM (containing 10% FBS)), and 1.5 ml of medium-diluted Coptidis rhizome was added to each well Alkali, placed in a cell culture incubator (37°C, 5% CO 2 ) for 6 h, and the control group was incubated with DMSO of the same volume as coptisine in the experimental group for 6 h. Afterwards, the cells were taken out from the incubator, the medium was discarded, and washed twice with PBS. Add 500 μL of 0.25% trypsin (containing EDTA) to each well to digest for 2-5 min. Then, 500 μL of DMEM (containing 10% FBS) was added to each well to stop the digestion, and the cells were collected and...
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