Construction method and application of glioma cell line suitable for two-photon living imaging
A glioma cell and in vivo imaging technology, which is applied in the construction and application of two-photon in vivo imaging glioma cell lines, to achieve the effect of improving accuracy
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Embodiment 1
[0036] Construction of a glioma cell line (GL261-ZsGreen) expressing green fluorescence:
[0037] Step 1. Spread 5×10 cells in a 24-well culture plate 12 hours in advance 4 GL261 glioma cells, after the cell confluency reaches about 60%, add 6 μg / ml polybrene transfer agent to the DMEM medium, and then use ZsGreen fluorescence with CMV promoter and two-photon excitation band The pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro lentivirus with reporter gene and puromycin resistance gene was used to infect the pre-laid GL261 glioma cells;
[0038] Step 2. After the above-mentioned GL261 glioma cells infected with the lentivirus were grown for 48 hours, replace with fresh DMEM medium containing puromycin at a concentration of 2 μg / ml, and screen the GL261 glioma cells. Replace with fresh medium containing puromycin once every day;
[0039] Step 3. The glioma cells with green fluorescent label (GL261-ZsGreen) screened in step 2 are monoclonally amplified and stored frozen.
[0040] Construct...
Embodiment 2
[0046] Construction of a glioma cell line (GL261-ZsGreen) expressing green fluorescence:
[0047] Step 1. Spread 5×10 cells in a 24-well culture plate 12 hours in advance 4 A GL261 glioma cell, after the cell confluency reaches about 60%, add 7 μg / ml polybrene transfer agent to the DMEM medium, and then use ZsGreen fluorescence with CMV promoter and two-photon excitation band The pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro lentivirus with reporter gene and puromycin resistance gene was used to infect the pre-laid GL261 glioma cells;
[0048] Step 2. After the above-mentioned GL261 glioma cells infected with lentivirus were grown for 48 hours, fresh DMEM medium containing puromycin at a concentration of 2 μg / ml was replaced to screen GL261 glioma cells. Replace with fresh medium containing puromycin once every day;
[0049] Step 3. The glioma cells with green fluorescent label (GL261-ZsGreen) screened in step 2 are monoclonally amplified and stored frozen.
Embodiment 3
[0051] Construction of a glioma cell line (GL261-ZsGreen) expressing green fluorescence:
[0052] Step 1. Spread 5×10 cells in a 24-well culture plate 12 hours in advance 4 GL261 glioma cells, after the cell confluency reaches about 60%, add 8 μg / ml polybrene transfer agent to the DMEM medium, and then use ZsGreen fluorescence with CMV promoter and two-photon excitation band The pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro lentivirus with reporter gene and puromycin resistance gene was used to infect the pre-laid GL261 glioma cells;
[0053] Step 2. After the above-mentioned GL261 glioma cells infected with lentivirus were grown for 48 hours, fresh DMEM medium containing puromycin concentration of 3 μg / ml was replaced to screen GL261 glioma cells. Replace with fresh medium containing puromycin once every day;
[0054] Step 3. The glioma cells with green fluorescent label (GL261-ZsGreen) screened in step 2 are monoclonally amplified and stored frozen.
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