In vitro purification of self-sanguifacient dry cell and kit
A technology of hematopoietic stem cells and kits, which is applied in the field of biomedicine and can solve problems such as application limitations
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Embodiment I
[0044] Example 1 Ad-GFP measures the transfection efficiency of hematopoietic stem cells, breast cancer cells, and multiple myeloma cells and counts hematopoietic stem cells PBSC, breast cancer cells MCF-7 and MDA-MB-231, and multiple myeloma cells SKO-007 cells to 10 5 Each dish was inoculated in a 35mm dish, and 500 μl of serum-free 1640 culture medium (purchased from GIBCO, USA) was added to each dish. Add Ad-GFP to the above cell culture dish at a titer of 0, 25MOI, 50MOI, 100MOI, 200MOI, 400MOI, 800MOI, mix and shake at 37°C for 2 hours, add 500μl of 1640 culture solution containing 20% fetal bovine serum, set into 37°C CO 2 After 48 hours in the incubator, the Ad-GFP infection rate was counted under a fluorescent microscope and detected by flow cytometry. The results show that under the fluorescence microscope, MCF-7, MDA-MB-231, and SKO-007 can obtain 100% transfection at an Ad-GFP 100MOI titer, while for PBSC, even if the Ad-GFP infection titer is as high as 800MOI...
Embodiment II
[0045] Example II Evaluation of CCTCC98003's Killing Effect on Breast Cancer Cells and Multiple Myeloma Cells
[0046] 8×10 per well 3 Cells, inoculate MCF-7, MDA-MB-231, SKO-007 cells in 96-well plates. Add 100 μl of serum-free 1640 culture medium to each well, and transfect Ad-GFP, CCTCC98003 at titers of 0, 25 MOI, 50 MOI, 100 MOI, and 200 MOI respectively, and Ad-GFP was used as a control. Shake and mix in a 37°C incubator for 2 hours, add 100 μl of 1640 culture solution containing 20% fetal bovine serum, and place in a 37°C CO 2 After culturing in an incubator, the MTT assay was carried out after 96 hours. MTT detection was performed according to the conventional method, and finally the cell survival rate of different infection titers was calculated. See below for cell viability formula. Draw the dose-cell viability curve, use the least squares method to fit the curve, and obtain the IC for inhibiting 50% cell survival according to the obtained formula 50 . Cell s...
Embodiment III
[0048] Example III Effect of CCTCC98003 on CFU-GM Formation Ability of Hematopoietic Stem Cells
[0049] Freshly isolated PBSC cells were seeded in 24-well plates with 2000 CD34+ cells per well, added 200d1640, and were transfected with CCTCC98003 and Ad-GFP at 0, 100MOI, 200MOI, 400MOI, 600MOI, 800MOI, and 1500MOI, respectively. Mix and shake at 37°C for 2 hours, add 400 μl of semi-solid CFU-GM culture medium (purchased from STEM CELL, USA), mix well and place in 37°C CO 2 After 14 days of culture in the incubator, when macroscopic colonies appeared in the culture dish, the culture was terminated, and the number of CFU-GM colonies was counted under a microscope. The colony formation rate was calculated according to the following formula. Colony formation rate = number of colonies / number of seeded cells × 100%
[0050] The results showed that when CCTCC98003 was less than 800 MOI, it had no significant effect on the CFU-GM formation ability of hematopoietic stem cells. se...
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