Adipogenic Induction Kit For Human Mesenchymal Stem Cells

(1) Induction solution B: melt the additive SOLUTION B at 2-8℃ overnight, and rewarm the solution B at 37℃ for 30 minutes before use, and mix well. The basal medium A was divided into two equal parts, each 45 ml, and the additive B was added to prepare the complete induction medium B, which was fully mixed.
(2) Induction medium C: Melt solution C overnight at 2-8℃, add another 45 mL of basic medium A to prepare complete induction medium C, and mix well.
Note: The additive B must be fully reheated and the contents thawed sufficiently to be transparent without precipitation. The mixture into the induction complete medium must be well mixed and stored at 2-8 ℃. The whole process must be aseptic, with surfaces wiped with 75% alcohol before each reagent is opened.
(3) Oil red O dye solution (working solution) : take oil red O storage solution, mix it with distilled water in a ratio of 3:2 and filter it with neutral filter paper, that is, to prepare oil red O dye solution (working solution). The oil red O staining solution is ready for use. 2. Petri dish treatment:
Add appropriate amount of the coating solution D to the culture vessel, gently shake it to cover the whole bottom of the culture vessel, and let it stand at room temperature for 30-60 minutes. Discard the solution and let dry at room temperature.
Note: The coating solution C must be reheated at room temperature before use. It is recommended to use a six-well plate and add 1 ml of liquid D to each well. Stability of the results should be considered when using other culture vessels. The whole process should be operated in a super clean workbench to avoid contamination.
3, self prepared reagent:
(1) Digestive fluid (0.25% trypsin-0.04% EDTA or other)
(2) Phosphate buffer (DPBS)
(3) MSC complete medium
(4) 4% neutral paraformaldehyde solution
Note: < span style = "max-width: 100%; clear: both; min-height: 1em; MSC digestion is extremely easy to overdo. It is recommended to dilute 0.25% trypsin digestion solution twice and strictly control the digestion time.
1. Prepare the required MSC to be induced and differentiated. When the cell fusion reached about 85%, the cells were digested with digestive juice and resuscated with MSC complete medium.
2, according to 3× 10⁴ cells/cm2 density The cells were inoculated in the coated six-well plates with 2 mL MSC complete medium per well and cultured in a 5%CO< SUB >2 incubator at 37℃. < p style = "max-width: 100%; clear: both; clear: both; min-height: 1em; Or the inoculation density is low, until the cells grow to the next required density induction.
3. When the cell fusion reached 90%, the culture supernatant was discarded, induction solution B 2 mL/well was added to each well, and 5%CO< SUB >2 was cultured at 37℃.
< p style = "margin-bottom: 0pt; margin-bottom: 0pt; margin-bottom: 0pt; margin-bottom: 0pt; margin-bottom: 0pt; Add medium gently, slowly along the side wall of the vessel to avoid blowing up cells. The degree of fusion at the initiation of induction is critical to the induction effect. It is necessary to ensure that the cells have grown sufficiently dense before induction.
4. After 72 hours of induction culture, the original culture supernatant was discarded, induction solution C 2 mL/well was added, and the culture was continued at 37℃ with 5%CO₂.
5. After 24 hours of induction culture, discard the original medium, add induction solution B 2 ml/ well, and continue the culture at 37℃ with 5%CO₂.
6. Repeat steps 4 and 5 for 3-5 times, and replace with induction solution C when obvious lipid droplets appear in cells. < p style = "max-width: 100%; clear: both; clear: both; min-height: 1em; Cells in good condition can be cultured within 10 days. If no lipid droplet structure is found after 10 days, please pay attention to the analysis of the procedure and other reasons.
8. Replace fresh induction solution C every 2 days and continue culture until the fat drops are large enough.
< span style = "box-sizing: border-box; color: RGB (74, 74, 74); line-height: 22px; font-size: 14px! Important; white-space: inherit! Important;" The end time of culture should be selected according to the experimental needs. If the fat-forming area is too large or the fat drops are too large, which leads to the continuation of slices, it is not conducive to the presentation of results, so it is suggested to terminate the culture in advance. When MSC was induced in different laboratories, the time of lipid droplet appeared was different. If lipid droplet appeared in large area earlier, induction solution C could be replaced in advance.
9. At the end of induction culture, the culture supernatant was discarded, the cells were washed twice with DPBS, and the cells were fixed with 4% neutral paraformaldehyde solution at room temperature for 20 minutes.
< p style = "max-width: 100%; clear: both; min-height: 1em;
10. Discard fixation solution, wash twice with DPBS, add oil red O dye (working solution) 1 ml/ well and stain for 15 minutes.
< span style = "box-sizing: border-box; color: RGB (50, 50, 50); font-size: 14px! Important; word-break: break-word! Important;" If you want to get beautiful pictures, the dyeing time should be mastered by yourself.
11. Discard the oil red O dye (working liquid) and wash it with DPBS for 3 times.
< p style = "margin-bottom: 0pt; margin-bottom: 0pt; margin-bottom: 0pt; margin-bottom: 0pt; Use the dye solution as you go. After dyeing, the naked eye can see obvious red in the hole. During the dyeing process, it is necessary to avoid the air drying of tissue cells, which will affect the microscopic observation effect.
12, look under a microscope and take photos.
< p style = "max-width: 100%; clear: both; min-height: 1em;
13. Result determination: after the procedure was completed, a large area of red staining points could be observed under low power microscope, and red spheres could be observed inside the cells under 20 and 40 times microscope. The red coloring spheres were lipid droplets, indicating that MSC used in the experiment had lipogenic ability. Otherwise, the MSC used has no lipid capacity.
< p style = "max-width: 100%; clear: both; min-height: 1em;