I am trying to isolate Bone Marrow Mesenchymal Stem Cells (BM MSCs) from BM samples. My first attempt to acquire BM MSCs was based on the retrieval of BM Mononuclear Cells from the BM sample using a gradient centrifugation (Histopaque 1077, Sigma). It also entailed the use of RosetteSep™ Human Mesenchymal Stem Cell Enrichment Cocktail. A washing step containing Red Blood Cells Lysis Buffer with NH4Cl was also included. When I isolated the buffy coat and performed cell count the yield of the mononuclear cells was not very satisfactory. The BM sample that was given to me was 1,5 ml.
http://www.stemcell.com/~/media/Technical%20Resources/3/4/9/0/2/28578%20PIS100pdf.pdf?la=en
I recommend to dilute the buffy coat at least 1:4 with PBS. We had the same problem and increased the yoield a lot!
You can try a Leucosep filter tube. With your volume a 15 ml tube should be enough. You need to filter first the diluted BM through a 100 um strainer to remove small clots.
Thank you very much for your interesting input. I will try to implement your advices on my next isolation. The Rosette Sep is a negative selection protocoll. Unwanted cells (Rosetted, details are contained in the file I have attached) are brought down along with the RBCs during the gradient centrifugation.
I agree with Luis Vidal Conde. I usually dilute the BM samples 1:1 in PBS 10% ACD v/v. Other factor that may alter the MC yeld is the ficol/sample proportion that vary according to the sample volume. To a volume of 1.5 ml of BM I add 1.5 ml of PBS 10% ACD (1:1 dillution) and place the resultant 3ml on top of 2 ml of ficoll and perform the centriffugation and 3 washing steps in PBS 10% ACD. The ACD helps to keep the cells vianle during the separation process.
First, separate your other cells from RBC by using ACK lysis buffer. Then try to isolate your interested cell type. It will work fine.
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