Single Cell Sampling Using Laser Capture Microdissection: A Technique to Harvest Target Cells from a Heterogeneous Cell Population

Published: April 30, 2023

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 obtaining a single cell from a heterogeneous cell population using laser capture microdissection. The single isolated cell can be used for further genomic studies or cancer research.

Protocol

1. Single-cell Sampling Laser Microdissection ​NOTE: Single cells will be added to 4.5 µL of cell lysis master mix (components of WGA kit, Table of Materials and Reagents) for WGA. Prepare the cell lysis master mix according to Czyż et al. by mixing 5.0 µL of reaction buffer, 1.3 µL of octylphenoxypolyethoxyethanol (10% solution), 1.3 µL of 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethyl…

Açıklamalar

The authors have nothing to disclose.

Materials

McCoy's 5A Medium (1x) suppl. with L-Glutamine Thermo Fisher Scientific 16600-082 Culture medium used for HT-29 cells, adapt culture medium to cell line used for the experiments
HEPES Buffer Solution (1 M) PAA S11-001 Supplement for McCoy's 5A Medium
Fetal Bovine Serum Gold PAA A15-151 Supplement for McCoy's 5a Modified Medium
Penicillin-Streptomycin (100x) Biowest L0018-100 Supplement for McCoy's 5a Modified Medium
Phosphate Buffered Saline (1x PBS) Thermo Fisher Scientific 10010-015
Accutase Biowest L0950-100 Cell detachment solution (cell culture)
Water bath GFL Type 1003 Used for prewarming solutions
Incubator Thermo Fisher Scientific Used to incubate cells (cell culture)
50 mL reaction tubes VWR 525-0403
Roller mixer Stuart Scientific SRT6D Used to attach cells to the wire while keeping the cells from sedimenting
CellTrace CFSE Cell Proliferation Kit Thermo Fisher Scientific C34554 Used to label living cells (cytoplasmic label)
Hoechst 33342 H3570 Thermo Fisher Scientific Used to stain DNA (nucleic counterstain)
Centrifuge (equipped for holding 15 mL and 50 mL reaction tubes) Thermo Fisher Scientific Heraeus Megafuge 40R Used for pelleting cells
Observer Z1 (Fluorescence/laser microdissection microscope) Carl Zeiss Microimaging Inverted (immunofluorescence) microscope capable of laser microdissection; Fluorescence microscopes must be able to detect Hoechst 33342 (DAPI filter) and CFSE (FITC filter)
Bovine Serum Albumin (BSA) Sigma-Aldrich A4503-50G Used for coating glass/plastic surfaces
MS1 Minishaker Sigma-Aldrich Z404039 Used for vortexing
Detektor CANCER03 DC03 Gilupi GIL003 Functionalized wire capable of isolating and detaching EpCAMpositive cells (also referred to as catch&release, C&R)
Syringe needles (G20) VWR 613-0554 Used to puncture rubber caps in order to allow the non-functional part of the C&R to lead through it
Neubauer chamber Roth T729.1 Used to count 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
Delfia PlateShaker Perkin Elmer 1296-003 Used for agitation during cell detachment
1.5 mL tubes Eppendorf 30,125,150
Ampli1 WGA Kit Silicon Biosystems To be ordered via Silicon Biosystems
0.2 mL PCR tubes Biozym 710920
Cytocentrifuge and equippment Hettich Universal 32 Use cytocentrifuge at hand
Thermo cycler Bio-Rad DNA Engine Dyad Every thermo cycler with heated lid can be used
Microcentrifuge Roth CX73.1 Use desk-top centrifuge at hand
UV Stratalinker 1800 Stratagene 400072 Use DNA cross linker at hand

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Bu Makaleden Alıntı Yapın
Single Cell Sampling Using Laser Capture Microdissection: A Technique to Harvest Target Cells from a Heterogeneous Cell Population. J. Vis. Exp. (Pending Publication), e20397, doi: (2023).

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