Melanoma Stem Cell Isolation: A Method to Obtain CD133 Positive and Negative Melanoma Cells Using Magnetic-activated Cell Sorting
Published: April 30, 2023
Abstract
Source: Welte, Y. et al. Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma. J. Vis. Exp. (2013)
This video describes the technique of isolating CD133 negative and positive melanoma stem cells from a primary melanoma cell line using magnetic-activated cell sorting. The CD133 positive melanoma CSCs are further studied to understand the mechanisms of melanoma tumorigenesis.
Protocol
1. Magnetic Cell Sorting of CD133+ and CD133- Melanoma Cells Using LS Columns Prepare MACS buffer containing 2 mM EDTA and 5% FCS in PBS. Mix by inverting and sterile-filtrate buffer using a 0.22 μm Steritop-GP Filter Unit. Chill buffer to 4-8 °C and degas before use. Pre-warm the Accutase, PBS, and Quantum 263 medium to 37 °C. Wash 80% confluent cells with PBS. Add an appropriate amount of Accutase and incubate until cells de…
開示
The authors have nothing to disclose.
Materials
| Accutase | PAA Laboratories GmbH | L11-007 | |
| Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) | Sigma-Aldrich, Inc. | E-7889 | |
| CD133/1 (W6B3C1) | Miltenyi Biotec GmbH | 130-092-395 | |
| Indirect CD133 Micro-Bead Kit human | Miltenyi Biotec GmbH | 130-091-895 | Includes: CD133/1(AC133)-Biotin, anti-Biotin MicroBeads and FcR Blocking Reagent |
| Quantum 263 | PAA Laboratories GmbH | U15-815 | |
| MACS Separator Multi Stand | Miltenyi Biotec GmbH | 130-042-303 | |
| MACS Seperation Columns 25 LS Columns | Miltenyi Biotec GmbH | 130-042-401 | |
| QuadroMACS Separator | Miltenyi Biotec GmbH | 130-090-976 |
