Any better reagent for isolation of PBMC from whole blood? - (May/14/2008 )

Hello
I am new in this forum,
I am working on the lymphoblasts cells.I derived them from the peripheral blood of the acute lymphoblastic leukemia patients using the same methad(Ficoll)
I have a little bit RBC contamination too but my big problem is that my cells slow down to grow and become smaller
Is there any growth factor that I should use?
could you please help me?I really need it
Thanks in advance
Acute

Here is what I do to isolate PBMCs, and it works very well (all materials should be a room temp!):

Tips:
- Use the collected blood as quickly as possible. These cells are not happy outside of the body they came from.
- Make sure all of your reagents are at room temperature before you start.
- Use a centrifuge on which you can turn the brake OFF.

1. Put blood into a 50 ml conical tube. Dilute 1:1 with sterile RPMI at room temp and mix it by pipetting up and down.
2. Put an equal volume of Ficoll (same volume as your blood, or a little more if you want) into a new conical tube. (Remember, your Ficoll should be at room temperature)
3. Slowly layer the blood/RPMI mixture over the Ficoll by slowly pipetting it down the side of the tube. Be careful not pipette too fast, or the blood with fall into the Ficoll.
4. Centrifuge the tube at 400xg (this is about 1600 RPM in our centrifuge, but it will vary of course) for 35 minutes at room temperature. TURN THE BRAKE OFF. If the brake is on, your interphase will fall into the other layers.
5. Toss the top plasma layer (yellowish). Collect the white interphase (your PBMCs), and put them into a new conical tube. Sucking up some Ficoll (the clear layer below the white ring of cells) is not a big deal. Bring the cells up to 50 mls in sterile, 1X PBS. Spin at 400xg for 5-10 minutes.
6. And there you have it, clean PBMCs. These cells require cytokines to survive. I use a cocktail from Stem Cell Technologies (IL-3, IL-6, Flt-3 and SCF), but you should look at what cytokines will maintain the cell types you're interested in.

Good luck!

Thanks a lot montana,
just one more question
should I use the equal volume of ficoll as my bloood volume or the RPMI/Blood Mixture volume?
Thnx in advance

I use an equal volume of Ficoll to blood (predilution). I usually get 10 mls of blood, so I dilute it with 10 mls of RPMI and the layer it on top of 10 mls of Ficoll.

Dear montana,
thanks alot it worked for me very well:)
just some questions....should I add cytokines to PBMC from the moment I isolated them or can I wait for 3 4 days untill their growth slow down?
how can I find which cytokine is best for lymphoblasts?cuz they are usually costly and I should pay it myself:(
what about insulin?I added insulin to one of my flasks it seems to make my cells happier.
tnx so much

I looked though papers on PubMed to see what other people were using for cytokines. I use a cytokine cocktail from StemCell Technologies. You're right, they are extremely expensive ($435 for 1 ml), but the cells with die without them. I've also looked into making up the cocktail myself, but the cost is about the same. Since I'm not sure exactly what you are trying to do, and because I don't know much about AML, I can't really offer any suggestions on what you should use. The cells might last for 24 hrs without cytokines in the media, but after that, mine start to look pretty pathetic and start to die.

As far as paying for the cytokines yourself... You really can't do these experiments without the right equipment. You should talk to you mentor about the cost of these materials and see if you can find funding through a training grant or something else. If you plan on using PBMCs frequently, you could easily spend $1000 a month on cytokines!

Dear montana,
thanks alot it worked for me very well:)
just some questions....should I add cytokines to PBMC from the moment I isolated them or can I wait for 3 4 days untill their growth slow down?
how can I find which cytokine is best for lymphoblasts?cuz they are usually costly and I should pay it myself:(
what about insulin?I added insulin to one of my flasks it seems to make my cells happier.
tnx so much