A method of promoting the growth of lung cancer cells
A lung cancer cell and cell technology, applied in the field of cell culture, can solve the problems of failure to contact, failure to contact tumor cell growth, failure to contact IL15, etc.
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Embodiment 1
[0043] Example 1: Preparation of CD45+CD215+ cells
[0044] The preparation method of the CD45+CD215+ positive cells is as follows:
[0045] 1) The third-generation immunodeficiency mouse NOD-scid-IL2Rγ- / - was killed by dislocation, and then soaked in 75% alcohol for 15 minutes for disinfection;
[0046] 2) After taking out the spleen of the mouse in a sterile environment, immerse it in 75% alcohol for disinfection and sterilization for 5 minutes;
[0047] 3) Take out the spleen and wash away excess 75% alcohol in sterile phosphate buffered saline (PBS);
[0048] 4) Place the spleen in a 100-mesh sieve, grind it in 3-5 mL of PBS, and prepare a single-cell suspension;
[0049] 5) Transfer the spleen cells to a centrifuge tube and centrifuge at 300g for 5 minutes;
[0050] 6) Discard the supernatant and add 3-5mL red blood cell lysate (eBioscience), and lyse at room temperature for 5 minutes;
[0051] 7) Add 5mL PBS to neutralize the erythrocyte lysate, and centrifuge at 300...
Embodiment 2
[0054] Example 2: Tumor cells co-cultured with CD45+CD215+ cells and IL15 cytokines
[0055] (1) The day before the co-culture, 30 tumor cells were planted in a 96-well plate and cultured overnight;
[0056] (2) CD45+CD215+ cells resuspended in a medium consisting of 1640 medium, 10% FBS by volume, 1% P / S by volume, and 20ng / mL GM-CSF, and the density was controlled at 2×10 4 / mL;
[0057] (3) Before culturing, select wells with 25-35 tumor cells in the wells for co-culture, and add 100 μL of resuspended CD45+CD215+ cells and 500 ng / mL of IL15 cytokine;
[0058] (4) At a temperature of 37°C, a humidity of 95% and CO 2 The culture was carried out at a concentration of 5% for 120 h, and the fresh medium was used for half replacement every 2 days during the culture period.
[0059] (5) After culturing for 120 hours, gently rinse the tumor cells with sterile PBS;
[0060] (6) Add 100 μL of CCK8 reagent, at a temperature of 37°C, a humidity of 95% and 5% CO 2 Incubate in the i...
Embodiment 3
[0062] Example 3: Tumor cells co-cultured with CD45+CD215+ cells and IL15 cytokines
[0063] (1) The day before the co-cultivation, 25 tumor cells were planted in a 96-well plate and cultured overnight;
[0064] (2) CD45+CD215+ cells resuspended in medium consisting of 1640 medium, volume ratio of 10% FBS, volume ratio of 1% P / S, and 20ng / mL GM-CSF, control density at 1×10 4 / mL;
[0065] (3) Before culturing, select wells with 25-35 tumor cells in the wells for co-culture, and add 120 μL of resuspended CD45+CD215+ cells and 50 ng / mL of IL15 cytokine;
[0066] (4) At a temperature of 35°C, a humidity of 90% and CO 2 The culture was carried out at a concentration of 2% for 150 h, and the fresh medium was used for half replacement every 4 days during the culture period.
[0067] (5) After culturing for 120 hours, gently rinse the tumor cells with sterile PBS;
[0068] (6) Add 100 μL of CCK8 reagent, at a temperature of 37°C, a humidity of 95% and 5% CO 2 Incubate in the inc...
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