G-10 Column Based Leukemia Cell Sorting: A Method to Purify Acute Lymphoblastic Leukemia Cells from Bone Marrow Stromal Cells

Abstract

Source: Slone, W. L.,et al. Modeling Chemotherapy Resistant Leukemia In Vitro. J. Vis. Exp. (2016).

This video describes the separation of acute lymphoblastic leukemia cells from bone marrow stromal cells using gel-type ten cross-linked dextran G-10 columns. This method helps recover the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells generating a "phase dim" tumor cell population.

Protocol

1. Loading Co-culture Cells onto G10 Column NOTE: Make sure stopcock is completely closed before adding media containing cells to G10 column. Also, each subpopulation must be ran over a separate G10 column so not to introduce any bias between populations in downstream analysis. Using a 1,000 µl pipette, add 1 ml of each cell subpopulation in pre-warmed media to a separate G10 column drop-wise. Ensure that the media containing the cells remains…

Materials

G10 sephadex beads	Sigma	G10120	Referred to in manuscript as gel type 10 cross-linked dextran particles
10 ml sterile syringe	BD	309604
1-way stopcocks	World Precision Instruments, Inc.	14054-10
Human Osteoblasts	PromoCell	C-12720	Human osteoblast were cultured according to the supplier’s recommendations.
Human Bone Morrow Stromal Cells	WVU Biospecimen Core		De-identified primary human leukemia and bone marrow stromal cells (BMSC) were provided by the Mary Babb Randolph Cancer Center (MBRCC) Biospecimen Processing Core and the West Virginia University Department of Pathology Tissue Bank. BMSC cultures were established as previously described (*)
0.05% Trypsin	Mediatech, Inc.	25-053-CI
REH	ATCC	ATCC-CRL-8286	REH cells were cultured according to the supplier’s recommendations and recommended media
Glass wool	Pyrex	3950
50 ml conical centrifuge tubes	World Wide Medical Products	41021039	Used as collection tubes
15 ml conical centrifuge tubes	World Wide Medical Products	41021037	Used for cell collection
Fetal Bovine Serum	Sigma	F6178
SD-1	DSMZ	ACC 366	SD-1 were cultured according to the supplier’s recommendations and recommended media
100 x 20 mm Cell Culture Dishes	Greiner Bio-One	664160