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 on top or within G10 pellet. Allow cells to incubate on G10 pellet for 20 min at RT.
NOTE: Stopcock remains closed for duration of incubation.
2. Collecting Leukemic Cells from G10 Column
- Add 1-3 ml pre-warmed media to each G10 column. Open stopcock valve and allow media to slowly exit the column drop-wise.
NOTE: It is crucial to maintain a slow flow rate from the column or the G10 pellet containing BMSC/OB can wash out of the column and contaminate the isolated leukemic cells. - Continue to add pre-warmed media in small increments (1-2 ml) to G10 column until a total of 15 to 20 ml has run through column and has been collected. Close stopcock valve and cap collection tube.
NOTE: If a G10 particle pellet is seen at the bottom of collection tube, gently remove media from the tube leaving G10 particle pellet undisturbed and transfer to new tube. - Centrifuge collected media at 400 x g for 7 min at RT. Remove supernatant and resuspend cell pellet in buffer appropriate for downstream application.
- Cells are now a pure population of leukemic cells free of BMSC or OB contamination and are ready to be applied to downstream applications at user discretion.
NOTE: Leukemic cell viability should remain unchanged when passing cells through G10 columns.
Açıklamalar
The authors have nothing to disclose.
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 |
