Single Cell Sampling Using Micromanipulation: A Technique to Isolate Single Cells from Cancer Cell Suspension

Abstract

Source: Chen S. et al. Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization. J. Vis. Exp. (2018)

This video describes the technique for isolating single cells from a pool of target cells to carry out downstream analysis. In this method, a micromanipulator fitted to a microscope is used for high-precision isolation of a single target cell.

Protocol

1. Single-cell Sampling

1. Micromanipulation
   ​NOTE: To allow downstream applications like whole-genome analysis (WGA), isolated single cells will be added to 2 µL of cell lysis master mix. Prepare a master mix according to the number of cells to be processed. Calculate extra volumes for loss due to pipetting and place cell lysis master mix on ice.
   1. Prepare cell lysis master mix (components of WGA kit, Table of Materials and Reagents). Briefly, mix 2.0 µL of reaction buffer, 1.3 µL of octylphenoxypolyethoxyethanol (10% solution), 1.3 µL of 4-(1,....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate Buffered Saline (1x PBS)	Thermo Fisher Scientific	10010-015
Vacutainer EDTA (or Na-heparin) tubes	BD	366450 (or 366480)	Used as reaction tube for ex vivo capture of target cells
Pasteur pipettes 150 mm	Volac	D810	Used for the low-volume application of cells to the C&R
Grease pen	Dako	S2002	Used on glass slides to hold cell suspension in place
Axiovert M200 equipped with Mikromanipulator MMJ and CellTram vario	Zeiss/Eppendorf		Use micromanipulator at hand
Microcapillaries (25 µm in diameter), CustomTip Type I	Eppendorf	930001201	Used for micromanipulating cells
0.2 mL PCR tubes	Biozym	710920