Establishing method of mouse bone marrow hematopoietic stem/progenitor cell in vitro senile cell model
An aging cell model and bone marrow hematopoiesis technology, applied in the fields of biotechnology and medicine, can solve the problems of difficult popularization, cumbersome operation steps, and long experimental cycle, and achieve the effects of simple operation, shortened modeling time, and difficult apoptosis
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Embodiment 1
[0025] Example 1 Primary isolation and in vitro culture of mouse bone marrow hematopoietic stem / progenitor cells
[0026] The primary isolation and in vitro culture of mouse bone marrow hematopoietic stem / progenitor cells were carried out according to the following steps:
[0027] 1) The mouse (4 weeks old) was killed by dislocation, and the mouse was sterilized with 75% alcohol, and the femur of the hind limb was removed by aseptic operation, and placed in buffer (containing 0.5% FBS, pH7.2, 2mM EDTA PBS, that is, the subsequent buffer ), remove the muscle;
[0028] 2) Aspirate the buffer with a 1mL syringe to flush the bone marrow. Pipette the cells with an unmarked soft pipette;
[0029] 3) Filter with a 30μm filter, centrifuge at 300g for 10min, and discard the supernatant;
[0030] 4) Add 1 mL of PBS to the precipitation, add 3 mL of erythrocyte lysate, mix well, and place at room temperature for 5 min. Mix by inverting several times during this time. Centrifuge at 3...
Embodiment 2
[0037] Example 2 Detection of Lineage by flow cytometry - c-kit + cell ratio
[0038] Check the purity of the sorted cells:
[0039] (1) Collect fresh Lin after sorting - c-kit + Cell and embodiment 1 cultivated Lin after 8 days - c-kit+ 1×10 cells each 6 Centrifuge at 1500rpm for 5min;
[0040] (2) Wash the cells once, add 10ul of CD117-PE, and incubate at 4 degrees for 15 minutes in the dark;
[0041] (3) Wash the cells once, dissolve the cell pellet to 200-500ulbuffer, and resuspend the cells. Flow cytometry (Becton Dickinso AccuriTM C6) detection. MACS Sorting Lin - CD117 + After detection of CD117 + The percentage of cells is (92.2%±4.5%, n=10, figure 2 A).
[0042] The cells were treated with stemspan SFEM (stemcell#09600) medium+10ng / ml IL3+10ng / ml IL6+30ng / ml SCF+100UI / ml penicillin, 100UI / ml streptomycin double antibody in 5% CO 2 CD117 was detected again after 8 days of cultivation in the incubator at 37°C + The percentage of cells is (86%±3.9%, n=10,...
Embodiment 3
[0043] Example 3 Senescence-associated β-galactosidase staining
[0044] Follow the steps below: Collect each group of 1×10 6 Add 1 ml of β-galactosidase staining fixative to each HSC, and fix at room temperature for 15 minutes. Wash with PBS, add β-galactosidase staining solution (Beyond C0602) and mix well, 37°C, CO free 2 Incubate overnight (16h) under the condition, examine under the microscope, and calculate the positive cell rate by the number of positive cells among the 400 cells randomly counted. SA-β-gal (senescence-associated β-galactosidase) is a hallmark indicator of aging. SA-β-gal staining positive cells increased in size, cytoplasm was blue, and negative cells were not stained. Our experimental results showed that the aging group ( image 3 B) The percentage of HSC SA-β-galactosidase positive cells was significantly higher than that of the young group ( image 3 A) HSC, the stained cells in the aging group almost covered the field of view, and the cells in ...
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