Method for utilizing malignant pleural effusion for separately culturing primary cancer cells
A technology for primary tumor cells and malignant pleural effusion, applied in tumor/cancer cells, cell dissociation methods, animal cells, etc. The effect of solving culture failure and optimizing culture conditions
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Embodiment 1
[0053] [Example 1] Isolation and culture of primary tumor cells from pleural effusion
[0054] (1) Preparation before the experiment: Add 10 mL of sample collection solution into a 50 mL sterile centrifuge tube to collect pleural effusion;
[0055] (2) Collect the patient's pleural effusion samples into the above centrifuge tubes, each tube can collect 30mL pleural effusion, collect 2 tubes from each patient, and transport them to the cell culture room under refrigerated conditions;
[0056] (3) Centrifuge at 1000rmp for 5min, discard the supernatant;
[0057] (4) Add 5 mL of erythrocyte lysate to remove red blood cells, mix gently, place at room temperature for 5 minutes, shake properly in the middle, centrifuge at 1000rmp, and discard the supernatant for 5 minutes;
[0058] (5) Add 15mL of PBS buffer to wash twice, centrifuge at 1000rmp, and discard the supernatant for 4min;
[0059] (6) Resuspend the cell pellet with 1mL of KL medium, inoculate the cells in a T25 culture ...
Embodiment 2
[0061] [Example 2] Subculture of primary tumor cells from pleural effusion
[0062] (1) When the primary tumor cells of pleural effusion cultured in T25 culture flask proliferate to an abundance of more than 85%, they can be digested and passaged, and the old medium is discarded;
[0063] (2) Rinse the cells with PBS buffer to remove cell secretions that hinder trypsin digestion, wash twice, and be gentle;
[0064] (3) Add 1ml, 0.05% trypsin-EDTA and incubate at 37°C for 2min, shake the cell culture flask lightly to make the digestion solution cover the entire cell layer, then pat;
[0065] (4) When 90% of the cells fall off from the culture flask, add 2 mL of PBS stop solution containing 10% fetal bovine serum to stop the digestion reaction;
[0066] (5) Transfer all the cell suspension that terminated the digestion to a 15mL centrifuge tube, rinse the culture bottle once with PBS buffer, and centrifuge to discard the supernatant;
[0067] (6) Resuspend the cells with 1mL o...
Embodiment 3
[0069] [Example 3] The growth curve drawing of primary tumor cells in pleural effusion
[0070] (1) When the primary tumor cells from pleural effusion cultured in T25 culture flasks proliferate to an abundance of more than 85%, they can be digested and centrifuged and planted in 96-well plates;
[0071] (2) When the primary tumor cells from pleural effusion digest the centrifuged seed plate, repeat the operation steps of Example 2 (2) to (5);
[0072] (3) Prepare a single cell suspension with KL medium, mix the trypan blue and the cell suspension 1:1 and measure the cell number and viability with a cell counter, inoculate 200 μL of the cell suspension in each well on a 96-well plate, and the number of cells is 6000 cells / well, set up 12 multiple holes per day, put in 37°C CO 2 Culture in the incubator for 1 to 7 days respectively;
[0073] (4) Remove the solution in the plate every day, and add 100 μL of serum-free DMEM medium containing 10 μL CKK-8 detection reagent to each...
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