Based on my experience here are some suggestions.

1. Would it be best to use human TNF-alpha or mouse TNF-alpha?

Incubation of differentiated 3T3 L1 adipocytes with Human TNF-alpha (10ng/ml) for 24h induces significant insulin resistance.

2. What is an appropriate buffer to prepare the stock solution of TNF-alpha?

TNF-alpha from Abcam is soluble in water.

3. If I aliquot and freeze the solution, how long can I keep it in -20C. And for an aliquot that I thaw and dilute, how long can it stay in a 4C fridge?

Make small aliquots and freeze them at -20C or -80C. Avoid freeze thaw. The solution can be stored in -20C or -80C for a long time. I have used aliquots frozen a year ago and they were fine.

4. For testing glucose uptake, can I use an indirect glucose assay (measuring how much glucose is left in the medium)? Or would you recommend using 2-NBDG?

The best way for measuring glucose uptake in adipocytes is by using 2-Deoxy-d-[2,6-3H]glucose. 2-NBDG works fine in cancer cells but I had little success in 3T3 L1 adipocytes. I am not sure whether indirect glucose assay (measuring how much glucose is left in the medium) will work in adipocytes.

Best,

Sameer

Hello Mr. Decha Sermwittayawong,

I totally agree with Sameer Mohammad ,

You can also try to consider this,

1. You can incubate 3T3-L1 adipocytes with mouse-TNF-alpha with various strategies

A. 4 ng/ml for 48 hrs by changing media by changing twice a day

B. 10 ng/ml for 72 hrs by replacing the media daily

2. You can make the main stock in water or the solvent prescribed by the company datasheet.

Further working solutions can be made by using Phosphate Buffer Saline(PBS).

3. Please try to save the main stock at -80 degrees for long-term use. For the short term, you can keep at -20 degrees and don't freeze-thaw & Try to make small aliquots as per requirements.

4. For Glucose Uptake, You can use 2-deoxyglucose uptake.

Here I am adding a link to the reference paper where they have made IR adipocytes by using TNFa and they have also performed the Glucose uptake assay.

• Selective Insulin Resistance in Adipocytes by Shi-Xiong Tan et al (PMID:- 25720492)

Thank You

To prevent adsorption of TNFa to the vessel surface and thus loose activity , I'd add a carrier protein like BSA or just use serum for the preparation of the stock solution.

Measuring the glucose concentration in the medium very likely won't be precise enough, as you have some variable which are difficult to control precisely, as cell number, growth rate, metabolic rate and the exact volume of the medium. Plus, the glucose concentration should not change much over time. Compare the weight of your cells with the amount of glucose in the medium. If you have access to a clinical analyzer, you might check though, if you can draw anything conclusive from glucose and lactate values.

Sameer Mohammad and Nilesh Khandelwal Thank you very much for your help. For storage of TNF-alpha, do I need to add some glycerol into water or PBS to mix with the protein to prevent protein denaturation after thawing? Thank you!

Wolfgang Schechinger, I also thank you for your suggestion. How much BSA do I need?

Decha Sermwittayawong BSA concentration is not critical. 1mg/ml to 5mg/ml usually works fine. In addition, you also could pre-treat the vials for the aliquots with the BSA solution, then their surfce has been ~ saturated. If you have the opportunity, then snap freeze the aliquots in liquid nitrogen to prevent crystal formation. Ideally, store then them at -80°C or liquid nitrogen, but for short time, -20 should do. Try to avoid adding glycerol, unless you are sure it works. Some proteins don't like it. Antibodies e.g. frequently are inactivated when the solution contains glycerol and solidifies. With aliquots, you don't really need that.

Wolfgang Schechinger Schechinger, thank you very much for your advice. The protein is very expensive and that's why I want to make sure that I don't make mistakes. I guess I have more questions to ask you. Let's say I use PBS to dissolve TNF-alpha and make aliquots as you suggested. When I use one aliquot, how long can I keep it in a 4C fridge? 2 weeks? 2 months? Thank you very much.

Decha Sermwittayawong biggest problem IMHO will be the risk of microbial contamination. So adding some antibiotics (the same that you will use in the media) might be useful. As you always will have some degradation over time (you might need an activity test to find out how long it is actually and reproducibly stable), preparing frozen aliquots of the amount needed might be ideal. Esp. when you need to dilute the TNFa, the carrier protein will prove very useful.

hello everyone, I have some issue in my experiment I’m using 3t3-l1 cells from last 4 months and until now I haven’t successfully induced adipogenesis in my cells. My protocol is Med1 pre-adipocytes medium (DMEM high glucose + Newborn calf serum 10%+ p/s 1%) supplemented for two days after confluency, then adipogenesis induction medium (DMEM high glucose + fbs 10% + Insulin 2ug/ml + dexamethasone 1uM + ibmx .5mm and p/s 1%) for 2 days, then med 3 (DMEM high glucose + fbs 10% + Insulin 2ug/ml + p/s 1%) until 14 days. I tried both human and bovine insulin but whenever I go for oil red o stanning no any change can be notice in preadipocytes and adipocyte cells as well as during the adipogenesis period I can’t see any changes or formation of lipid droplets in my cells. Recently I bought new cells but I am facing same problem with that too.

The problem might be that with DMEM high glucose, the cells already are in overdrive. Do you see a difference in glucose uptake upon stimulation?

Wolfgang Schechinger sir i passage cells on 80% confluncy and i cant see any difference in glucose uptake among the groups. could you please suggest me which manufacturer DMEM high glucose will be better

DMEM is so well known and there is no secret about it, I'd simply try the cheapest I could get. The issue IMHO is the high glucose. You could try the low glucose variety and also check the insulin content of the serum you're using (maybe you can ask a department of laboratory medicine for assistance, insulin determination is done as routine in hospitals).

Wolfgang Schechinger thank you sir for your kind suggestion i will try and will get in touch with you after results

Hi Nawaz Khan , based on my experiment, i found the insulin concetration is important in inducing adipogenesis. I firstly used 1 ug/ml insulin in induced-medium, the result is not good. Then I tried 10ug/ml insulin (the concentrations of IBMX and DEX are the same as yours), the adipogenesis is visible to naked eyes after 48 hours, but the cells' ability of adhering is weakened, so i treated them very very tenderly. Some researchers recomand 1-10ug/ml insulin. Maybe you can try higher insulin concentration, like 5 ug/ml or 10ug/ml. Wish you luck~