Reagents and materials:
Complete medium (CCM): α-MEM: α-low-limit basal medium containing glutamine, no nucleotide or deoxynucleotide; addition: 20% additional L-glutamine 2mmol/L, FBS The hybridoma was purified, non-heat inactivated, penicillin 100 U/ml, and streptomycin 100 μg/ml. Filtration sterilization. Store at 4 ° C for no more than 2 weeks;
BDM (bone differentiation medium): CCM contains 5 mmol/L β-glycerophosphate, 50 μg/ml ascorbic acid-2-phosphate and 1 nmol/L dexamethasone. Filter sterilization;
PBSA;
Trypsin/EDTA;
Polypropylene centrifuge tube, 15ml and 50ml;
Tissue culture dish, 6 wells, with a pore area of ​​9.6 cm 2 ;
ARS: 1% ARS was prepared in distilled water, pH was adjusted to 4.1 with 0.5N ammonium hydroxide, and filtered to remove bacteria;
Formalin buffer, 10%;
Improved Neubauer blood cell counter;
experimental method:
Collecting MSCs from monolayer cells;
2 ml of CCM was added to each well of a 6-well culture plate for a total of 1 x 104 cells (for human or rat). The final density is about 1 × 103 cells / cm 2;
Mark the upper 3 holes as “osteogenesis†and the lower 3 holes as “negativeâ€;
Incubate at 37 ° C, 5% CO 2 , and change the medium every two days until the cells reach 7.%-80% confluence;
When the desired cell density is reached, a complete prototype is aspirated from the well, 2 ml of BDM is added to the wells of the 6-well culture plate, and 2 ml of CCM is added to the lower row of wells;
Incubate at 37 ° C, 5% CO 2 and replace the medium every two days. Cells were stained on day 21 for ARS detection;
The differentiated MSCs were taken out from the incubator, and the monolayer cells in each well were washed twice with 2 ml of PBSA;
2 ml of formalin was added to each well, and monolayer cells were fixed at room temperature for 10 min;
The formalin was aspirated, and 2 ml of PBSA was added to each well for two times, and then washed with 2 ml of distilled water;
Add 2ml ARS solution and incubate for 30min at room temperature;
Rinse the wells with excess distilled water until the background staining in the "negative" wells is maximally cleared;
The degree of deep red was evaluated by observing the monolayer cells labeled "osteogenesis" with a microscope. When the osteogenic differentiation reached a satisfactory level, the ARS staining area of ​​the monolayer cells exceeded 50%. At this time, the monolayer cells can be stored for up to 7 days in 4 ° C water. Alternatively, the ARS can be extracted from the stained monolayer cells and assayed using the protocol previously described;
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