http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html Life Science Discussion en-us sci7feed@sci7.nojunkorherepleasespam.com hourly 1 2005-05-05T12:00+00:00 http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html I got a very good differentiation rate. I dont need to get fully mature adipocytes, because adipocyte can express the protein that i am looking for after 1 week of differentiation.
It could be great if I could use 6 well plate, but i don't know if i can use it with fluorescent microscope because of the well size.
Now, I am growing cells on coverslip and then I can perform immunostaining.
But i have a question :
My primary antibody is scFV monoclonal a...]]> Mon, 11 Oct 2010 16:04:00 GMT http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html I have worked extensively with differentiation of 3T3L1 to mature adipocytes.
3T3L1 differentiate very well in a petri dish. I used to work with six well plate. From the day of seeding, it takes 10 days for the 3T3L1 to differentiate into mature adipocytes. I will suggest that give media change every 48 hrs.
Do not trypsinise after differentiation.
I hope you can proceed with your immunostaining in a petri plate/6 well plate.
All the best...!!!!]]> Wed, 06 Oct 2010 06:06:44 GMT http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html The MID containing medium is removed on Day2, and changed into medium containing Insulin alone for another 2 days. Starting from Day4, you can use the regular culture medium without any supplements until the cells are ready...
Hope this helps and good luck.]]> Sun, 05 Sep 2010 22:05:42 GMT http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html Do you keep the cells with MID until differentiation or remove it after a couple days?
Thanks,
V.
]]> Sun, 05 Sep 2010 21:01:06 GMT http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html Yes, I did mean by visualizing directly under the microscope. Sorry for the confusion.
In my hands, the fat droplets start showing up on Day 2 and the number will increase gradually as the differentiation progresses. And I usually judge the differentiation via the distribution of the fat vacuoles- Good differentiation will give you pretty even distribution. You are right, it is really hard to tell individual cells, but the thing is one can't really get 100% diff...]]> Thu, 01 Jul 2010 20:58:50 GMT http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html Because when I used microscope, it is difficult to see if they have been differentiated because the confluency is 100% (1500X) and to hard to see how they look like individually.
]]> Thu, 01 Jul 2010 19:03:35 GMT http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html To prepare the differentiation medium, I usually prepare the MID stocks individually and add them into the culture medium before feeding the cells. Below is my protocol-
100x IMBX: 11.5 mg/ml in ddH2O + 1N KOH (1 drop or 2 so that it is totally dissolved)
1000x Insulin: dissolved in ddH2O to make 10mg/ml, then dilute in DMEM 1:10
1000x Dex: 390ug/ml in absolute Ethanol
I usually judge the differentiate by eyes instead of red oil staining... But the sta...]]> Thu, 01 Jul 2010 17:49:04 GMT http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html It would help me a lot.
About the differentiation, I am using IMBX 0.5mM, dexamethasone 1µM, insulin 10µg/ml.
To make up this media, did you add directly the components (in powder) to the basal media (DMEM high glucose 4.5g/l) before sterile filtration or did you dilute them before in solution and then you add the required volume ? because I am not sure (with the first method), I really differentiate my cells. I am scared about dexamethasone/IMBX/in...]]> Thu, 01 Jul 2010 14:54:04 GMT http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html I differentiated L1 adipocytes on the cell culture dishes (look a lot like petri dish, but treated for mammalian cell culture) instead of the flasks, and it works fine.
However, for your experiment purpose, I suggest you to grow/differentiate the cells in chamber slides (here is some selections), thus you can easily perform your immunofluorense staining and the downstream analysis.
Good luck.]]> Wed, 30 Jun 2010 16:32:03 GMT http://www.scientistsolutions.com/t14856-imunostaining+on+adipocyte.html Ok, I would like to grow 3T3 L1 and then differentiate them into adipocyte cells. After this, I will add an primary antibody (monoclonal scFV) to bind a membrane protein (specific membrane protein expressed by adipocyte cells) and secondary antibody (conjugated with FITC groupment).
The problem is that. I don't know if it would be possible to do the differentiation in a petri dish. I need my 3T3 L1 cells reach 100% of confluency before differentiate them. I cou...]]> Wed, 30 Jun 2010 16:10:06 GMT