Differentiation of 3T3 with IBMX, insulin, Rosiglitazone and

dexamethasone:

Data:
Start of the experiment: Day 02-03-2023
End of the experiment: Day 21-03-2023

Aim:
Learn to make a cell differentiation and observe the entire growth process of these
cells and determine if the different compounds have a great effect or not.

Material:

Plate 12 pouets (), Medium DMEM (10741574 (high glucose)) + 1%P/s (15140-122) + 10%
FBS(11573397), Giemsa (), Methanol (), Oil red, IBMX (), insulin (), Rosiglitazone ()
and dexamethasone (),glyphix diluted 0.5 ().

Method:

● Seed a P12 with 3T3 cells, from a 100% plate making a 1:4 dilution to
distribute among the 12 wells..

After 2 days of confluence start differentiation:

1. Remove the middle.

2. Put the different media in each well.
3. Cultivate for 2 days.
4. Remove the middle.
5. Put DMEM medium + 10% FBS + 1% P/S + 10ug/ml insulin per well.
6. Cultivate for 2 days.
7. Remove the middle.
 8. Put DMEM medium + 10% FBS + 1% P/S.
9. Cultivate and change the medium for 12 days.

Staining with oil red:

1. Remove the middle.

2. 2 washes with PBS.
3. Put 500ul of glyphix.
4. Incubate for 25 min at room temperature.
5. Put 500ul of oil red diluted 3/2.
6. Incubate for 2 hours.
7. Removethe oil red.
8. Do as many washes as necessary.

Results

Before differentiating them:

Control With everything +Rosiglitazone With everything -Rosiglitazone

After differentiating them:

Control With everything +Rosiglitazone With everything -Rosiglitazone

Conclusions:
It was observed that in the wells with rosiglitazone there is greater differentiation of
adipocytes, compared to thepouets where no rosiglitazone was added.
Before the microscope the control andpouets where no rosiglitazone was added.
They did not have much representative difference from one another, although there
were some more differentiated ones.