Stem cells from human exfoliated deciduous teeth (SHED)
Postnatal human dental pulp stem cells (DPSCs)
Reagents and materials:
1. PBS balanced salt solution (PBSA) without magnesium and calcium;
2. MSC medium: α-MEM containing 2mmol/L glutamine, adding 15% fetal bovine serum, 0.1mmol/L L-ascorbate phosphate, 100U/ml penicillin and 100μg/ml streptomycin;
3. Type I collagenase;
4. Neutral protease;
5. Reagents required for immunomagnetic bead sorting;
(1) PBSA with 1% BSA
(2) Primary antibody reactive with DPSC, using STRO-1 (mouse anti-human MSC; IgM), CC9 (mouse anti-human CD146/MUC-18; IgG2a), or 3G5 (mouse anti-human outer membrane cells; IgM)
(3) Goat anti-mouse IgG-binding or rat anti-mouse IgM-binding magnetic beads
6. Culture bottles or petri dishes;
7. Surgical blade and holder;
8. 70μm cell strainer;
9. Rotary mixer;
10. Dynal MPCR-1 Magnetic Separator
experimental method:
1. Cut the pulp tissue into small pieces with a surgical blade;
2. Place the shredded tissue in a collagenase/neutral protease mixed solution (1:1);
3. Incubate at 37 ° C for 30-60 min, intermittently shake the tissue / digestive enzyme mixture;
4. After digestion, add enough MSC medium to stop the enzyme activity;
5. The suspension was filtered through a 70 μm cell membrane to remove cell debris to obtain a single cell suspension;
6. Centrifuge at 500g for 6min;
7. Pour off the supernatant and resuspend the cells in MSC medium. Then performing immunomagnetic bead sorting;
(1) Incubation with primary antibodies reactive with DPSC cells, including STRO-1 (mouse anti-human MSC; IgM), CC9 (mouse anti-human CD146/MUC-18; IgG2a), or 3G5 (mouse against human) Outer membrane cells; IgM), 20 μg/ml, incubated on ice for 1 h;
(2) Wash twice with PBSA containing 1% BSA, centrifuge at 600g for 6min;
(3) Incubating with goat anti-mouse IgG-binding or rat anti-mouse IgM-binding magnetic beads, 4 °C rotary mixer for 40 min;
(4) separating the cells bound to the magnetic beads by a Dynal MPCR-1 magnetic separator according to the protocol recommended by the product manual;
8. Count the cells and inoculate them on a culture flask or dish at a density of (1 × 10) × 10 3 cells/cm 2 ;
9. Incubate in MSC medium, 37 ° C and 5% CO 2 incubator;
10. 7 days after cell separation, the flask was washed with PBSA and the medium was changed. The medium can then be changed twice a week until the cells reach confluence;
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