1. Where are the cells from?
The preadipocytes are isolated from human subcutaneous or visceral adipose tissue. The adipocytes are differentiated in vitro using these isolated preadipocytes.

2. How do you differentiate the cells?
The isolated preadipocytes are plated into the culture ware and treated with our Apipocyte Differentiation Medium (DM-2 or OM-DM) to stimulate adipocyte differentiation.

3. When do the cells differentiate?
Oil droplets should appear within 4-7 days after differentiation is induced. They look extremely small initially. Lipid accumulation continues throughout the first two weeks. The oil droplets gradually fuse to several big locules.

4. Can I pass the cells?
Adipocytes cannot be passed since they float after trypsinization. Preadipocytes can be trypsinized and replated several times. Preadipocytes grow slower with each passage and differentiate poorly after passage 4. Cells are shipped at Passage 2-4.

5. How fast do the cells replicate?
The average doubling time is 48-84 hours. However, keep in mind that the replication rate for human preadipocytes varies slightly from patient to patient.

6. How long do the cells last?
Adipocytes retain similar morphology and express adipocyte specific genes for at least 4 weeks after induction of differentiation. [NOTE: Cultured adipocytes are usually shipped at 2 weeks old.]

7. Is the beta adrenergic receptor present on these cells?
The subcutaneous adipocytes express beta-1 adrenoceptor and beta-2 adrenoceptor The mRNA for the Β receptor is not detectable by Northern analysis.

8. Do the cells express leptin? How do you measure it?
Yes. Mature adipocytes (greater than 2 weeks post differentiation) secrete 1-5 ng leptin from 50,000 cells in 24 hours. The leptin level stays the same for 5 - 6 weeks.

9. Do the cells respond to insulin treatment?
Yes. Insulin stimulates lipid accumulation within two days. Insulin stimulates both glucose uptake and phosphorylation of IRS1 within 20 minutes.

10. Can I differentiate the cells myself?
Yes. You can order preadipocytes and pre-made culture media for adipocyte differentiation. The protocol for differentiating the cells can be found in our manual or at our website ( Cell Manuals ).

11. Do you test for pathogens? How?
 Yes, Zen-Bio utilizes a third- party, FDA-certified testing lab to test all samples for HIV 1&2, HTLV 1&2, Hep B & Hep C.

12. Do the cells express the uncoupling proteins? Which ones?
The UCP1 mRNA is detected by PCR after stimulation with PPAR. agonist. UCP2 mRNA is expressed. We do not know if UCP3 is expressed or not.

13. Can I order visceral adipose?
Yes, we have cells from visceral depots available. Can I request cells from specific depots? Yes, we often have cells from various depots available. Please inquire as to price and availability.

14. How do I obtain RNA from the cells? How much RNA can I expect?
Use RNA tri-reagent (Sigma) or a guanidinium isothiocyanate solution (Chomcynzski protocol, reference number 4). You can expect approximately 20g total RNA from a 10 cm dish of confluent preadipocytes and 40-60g of RNA from a 10 cm dish of adipocytes.

15. What patient information do I get?
Donor's sex, age, and BMI will be provided.

16. Are the cells from one donor?
Yes. We can also provide lot numbers containing cells mixed from 5 to 8 donors to get average responses. Please inquire about availability of single donor and mixed donor lots (called a superlot) at the time the order is placed.