AdipoInducer Reagent (for animal cell)

Transcription

1 AdipoInducer Reagent (for animal cell) Product Manual

2 Table of Contents I. Description... 3 II. Kit components... 3 III. Materials Required but not Provided... 4 IV. Storage... 4 V. Protocol... 4 VI. Experimental example... 5 VII. Related products URL:

3 I. Description This reagent efficiently induces adipocyte differentiation in mouse embryonic fibroblast 3T3-L1 cells (ATCC No. CL-173), rat brown/white preadipocytes, mouse/rat/rabbit bone marrow cells and other animal cells. This kit contains three of the most frequently used adipocyte differentiation-inducers: insulin, 3-isobutyl-1-methylxanthine and dexamethasone. Adipose differentiation can be achieved by culturing cells in an appropriate medium containing these inducer reagents. Adipocyte differentiation is an area of scientific focus because adipose tissue plays an important role in energy homeostasis. Researchers are studying transcriptional regulation of adipocyte differentiation, and the role of epigenetic regulation. 3T3-L1 cells are one of the models used to study this process because they differentiate efficiently into adipocytes when induced by insulin, 3-isobutyl-1-methylxanthine and dexamethasone. II. Kit components (for 300 ml differentiation and 600 ml maintenance media) Kit contains 1 set (for 100ml differentiation medium ml maintenance medium) x 3 Additive reagents for differentiation medium (1)Insulin solution (final conc. 10 µg/ml)* 1 ml (2)Dexamethasone solution (final conc. 2.5 µm)* 0.5 ml (3)3-isobutyl-1-methylxanthine solution (final conc. 0.5 mm)* 0.1 ml Additive reagent for maintenance medium (4)Insulin solution (final conc. 10 µg/ml)* 1 ml x 2 *Final concentration after the specified volume is added to the medium x 3 This product is packaged in a box with its components arranged in the following manner. URL: 3

4 III. Materials Required but not Provided 1. Reagents 2. Materials - RPMI 1640 medium supplemented with glutamine [e.g., RPMI 1640 with L-Glutamine (Lonza Cat. #12-702F)] - Inactivated fetal bovine serum - Antibiotics (penicillin and streptomycin) [e.g., Penicillin-Streptomycin Mixture (Lonza Cat. #17-602E)] - 70% ethanol for disinfection - Sterilized pipettes IV. Storage - 20 V. Protocol - Dishes and multi-well plates for culturing adherent cells 1. Medium preparation A. Thaw additive reagents at room temperature or at 37 in an incubator. B. Disinfect the surfaces of medium bottles and additive-reagent vials by wiping or spraying with 70% ethanol. C. In a clean tissue culture hood, transfer 100-ml aliquots of an appropriate medium into each of two sterilized 100-ml bottles. Next, add the combination of inducers required for your experiment (additive reagents for differentiation medium (1), (2), (3)) to one of the bottles and 1 ml of the additive reagent for maintenance medium (4) to the other. Note: Additive reagent (3) is not essential for the differentiation of rat brown preadipocytes and mouse/rat/rabbit bone marrow cells. Media containing additive reagents may be stored at 4 for up to about 2 months. Smaller volumes of differentiation and maintenance media may be prepared. Adjust additive reagent quantities accordingly. Rat brown preadipocytes are susceptible to temperature change. Make sure to warm the medium to 37 before use. 2. Differentiation induction A. For mouse fibroblast 3T3-L1 cells 1) Prepare differentiation medium. (Add 100 µl of (1) insulin, 50 µl of (2) dexamethasone and 10 µl of (3) isobutyl-methylxanthine to 10 ml of serumsupplemented DMEM medium.) 2) Grow cells in growth medium until 2 days after cells reach 100% confluence. Remove growth medium. 3) Replace with differentiation medium and incubate for 48 hours (2 days). 4) Remove differentiation medium and replace with maintenance medium (add 100 µl of (4) insulin to 10 ml of serum-supplemented DMEM medium) and incubate for 5-10 days. Replace the medium every 5-7 days. B. For rat brown/white preadipocytes 1) Prepare 10 ml of differentiation medium. (Add 100 µl of (1) insulin and 50 µl of (2) dexamethasone to 10 ml of growth medium.) 4 URL:

5 Adipolnducer Reagent 2) Grow cells in growth medium to 90-95% confluence. Remove growth medium. 3) Add differentiation medium and incubate for 48 hours (2 days). 4) Remove the differentiation medium and replace with maintenance medium (add 100 µl of (4) insulin to 10 ml of growth medium) and incubate for 5-10 days. Replace the medium every 5-7 days. C. For mouse/rat/rabbit bone marrow cells 1) Prepare differentiation medium. (Add 100 µl of (1) insulin and 50 µl of (2) dexamethasone to 10 ml of serum-supplemented RPMI 1640 medium.) 2) Seed cells and incubate for 7 days. Remove growth medium. 3) Replace with differentiation medium and incubate for 48 hours (2 days). 4) Remove the differentiation medium and replace with maintenance medium (add 100 µl of (4) insulin to 10 ml of serum-supplemented RPMI 1640 medium) and incubate for 2-3 weeks. VI. Experimental example Rat brown preadipocytes were grown and induced to differentiate with differentiation media containing only (1) insulin, (1) insulin + (2) dexamethasone, or (1) insulin + (2) dexamethasone + (3) isobutyl-methylxanthine. After the differentiation medium was removed and replaced with maintenance medium, cells were observed on Days 1, 3 and 4. Microscopic observation revealed that brown preadipocytes were initially filled with small oil droplets and then differentiated into multilocular brown adipocytes. (1) (1) + (2) (1) + (2) + (3) Day 1 Day 3 Day 4 URL: 5

6 VII. Related products Osteoblast-Inducer Reagent (for animal cell) (Cat. #MK430) NOTE : This product is for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals. Also, do not use this product as food, cosmetic, or household item, etc. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC. If you require licenses for other use, please contact us by phone at or from our website at 6 URL: