Cell culture and adipocyte differentiation

Current protocol: Cell culture and adipocyte differentiation                                  

Materials and Reagents

1. Murine 3T3-F442A, C3H10T1/2, white subcutaneous (9W), or brown (9B) preadipocytes

2. 20 nM insulin (Sigma-Aldrich, catalog number: I6634)                               

3. 1 nM triiodothyronine (T3) (Sigma-Aldrich, catalog number: T2877)                               

4. 0.5 mM 3-Isobutyl-1-methylxanthine (Sigma-Aldrich, catalog number: I5879)    

5. 1 μM dexamethasone (Sigma-Aldrich, catalog number: D4902)                           

6. 0.125 mM indomethacin (Sigma-Aldrich, catalog number: I7378)                                    

7. 2.8 μM rosiglitazone (Sigma-Aldrich, catalog number: R2408)                             

8. Dulbecco’s modified Eagle’s medium (high glucose) (Life Technologies, Invitrogen™, catalog number: 11965)                         

9. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: 12657-029)           

10. Pen/Strep (Thermo Fisher Scientific, catalog number: 15140122)                                  

Equipment                              

1. 37 °C, 5% CO₂ incubator                            

2. Water bath                         

Procedure                               

Notes:                         

Pre-warm DMEM media (see Recipes) to 37 °C prior to the experiment                              

A. Seed the cells                                  

1. Seed 1 x 10⁵ preadipocytes in a 12 well plate and cultivate in DMEM media (see Recipes) until 90% confluence                                 

2. Change the media every 2 days                             

B. Adipocyte differentiation  (pro-browning stimulus)                            

Day 0:   1. Once the cells reach 90% confluence, change the media and cultivate for 2 days

Day 2   2. Replace the media for Differentiation media day 2 (see Recipes) and cultivate for 2 days                     

Day 4   3. Remove the media and replace with Differentiation media day 4 (see Recipes)

Day 6   4. After 2 days, replace the media with Differentiation media day 6 (see Recipes)

Day 8   5. Cells are fully differentiated and ready to use                   

Recipes                                   

1. DMEM media (supplemented with 10% FBS and 1% of Pen/Strep)                                  

2. Differentiation media day 2: DMEM media further supplemented with Insulin, Dexamethasone, IBMX, T3, Indomethacin, Rosiglitazone  (final concentrations in Material and Reagents)                              

3. Differentiation media day 4: DMEM media further supplemented with Insulin, T3 and Rosiglitazone  (final concentrations in Material and Reagents)                              

4. Differentiation media day 6: DMEM media further supplemented with Insulin and Rosiglitazone  (final concentrations in Material and Reagents)       

Acknowledgments                             

We are grateful for the technical assistance of E. E. Saviani and  for the supported by grants from FAPESP (2017/01184-9, 2017/07975-8, 2016/12294-7, 2010/52557-0, 2016/24163-4, 2016/23142-3, 2018/15313-8, and 2015/01316-7), CNPq (474397/2011-4 and 305069/2015-2), CAPES (1431744/2014-2016), and the Ecole Polytechnique Fédérale de Lausanne. INFABiC is co-funded by FAPESP and CNPq.                          

Representative images

Figure 1. Representative microscopic images of 9W preadipocytes before and after differentiation. Image of 9W preadipocytes at day 0 - 100% confluence and at day 8 - fully differentiated adipocytes.