yes , If a small number of cells necessary to complications by cell culture

can you isolation cml stem cell by positive selection or by removal of lineage-marker-positive (Linþ) cells

the marker can you using (CD34 + CD38 −)

Suspension cultures

Placed cells that have been collected into tubes containing serum-free medium,which consisted insulin and transferrin with 200 ml or

2 ml, of serum-free Iscove’s medium (SFM) containing

bovine serum albumin, insulin and transferrin ,

40 mg/ml low-density lipoproteins, 104M 2-mercaptoethanol 

and the and with or without the following combination

interleukin (IL)-3 (20 ng/mL)

 IL-6 (20 ng/mL)

G-CSF (20 ng/mL)

flt3-ligand(100 ng/mL)

 and Steel factor(100 ng/mL). The media were

replaced every week with fresh media with or without the same

imatinib and growth factor supplements.

and you need determine viable cell counts by hemocytometer counts .

see this Ref  :

( Instability of BCR – ABL Gene in Primary and CulturedChronic Myeloid Leukemia Stem Cells)

n Jiang , Kyi Min Saw , Allen Eaves , Connie Eaves

Shukran Saleh, thank you very much.

I have isolated CD34+ stem cells from blood of CML patients. At the time point of new diagnosis, you will get approx. 1-3% CD34+ cells in chronic phase. 20 ml of blood was sufficient to get 300.000-1.000.000 cells, sometimes even more cells. I would recommend you to go for CD34+ cell isolation using the Miltenyi system as compared to Robosep from Stemcell Technologies the recovery rate was higher with a comparable purity.

I don't know for which downstream application you want to have the RNA, I was even going for Affymetrix gene arrays by using an amplification kit, and just for going for some real-time PCRs, the amount of RNA should be more than sufficient. You might look into my 2 Leukemia-Publications. In one term we even went for CD34+ stem cell subpopulations by FACS sorting.

Furthermore, I found out that at least in the first week of patients treatment with Glivec (Imatinib) the overall number of cells did not decrease much.

After a successfull treatment over some time you will hardly be able to isolate CD34+ stem cells from blood, as the number will decrease to 0.1% or lower amount.

In case of a beginning resistance against a tyrosine-kinase-inhibitor, at first the BCR-ABL amount will increase before the cells are swamped out into the blood.

I hope that this information will help you a bit to plan your experiments.