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…

Divulgaciones

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

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Citar este artículo
Melanoma Stem Cell Isolation: A Method to Obtain CD133 Positive and Negative Melanoma Cells Using Magnetic-activated Cell Sorting. J. Vis. Exp. (Pending Publication), e20355, doi: (2023).

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