We found that all our myeloma samples were negative when we observed 13qS319, 13qS34, p53, t(11:14), t(4:14) and t(14:16) with FISH. Then we tried to sort CD138+ MM cells with FacS Aria III and afterward applied to the FISH. However, we saw that we lost our cells too much.
Could anyone suggest about our methodology or any other alternative methods?
Instead of going for cell sorting by flow cytometer you might try with immomagnetic staining and then magnetic isolation using MACS technology from Miltenyi. Depending on the purity you want to reach you can go for 1 or 2 column steps to increase the purity but you will of course lose more cells with 2 colums. I never did CD138 enrichment but enrichment of CD34+ cells (AML, CML, MDS als well as MM) and this was working out with a very good efficiency...
I agree with Daniela. However, be aware that CD138 is not so stable overtime and cells will loose CD138 by shedding or when in apoptosis (AnnexinV positive). Some proposed to use other markers such as CD319 or CD269 .(http://www.ncbi.nlm.nih.gov/pubmed/24385542?dopt=Citation)
Another option is to prepare cytospins and simultaneously conduct cytoplasmic immunoglobulin (cIg) staining and FISH.
You may want to look at publications by Tian E using a technique called TRI-FISH.
It is basically what Sabine mentioned a cytoplasmic Ig stain and then any other target of your liking.
We use it on a daily basis for patient sample here at the Myeloma Institute in Little Rock with excellent results.
I will try to sort MM cells by CD319 and then apply to TRI-FISH. Thank you
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