Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood

Protocol

NOTE: This protocol describes the isolation of HT-29 cells (human colon cancer cell line) from PBS or from artificial mixtures of HT-29 cells and peripheral blood. The same experiment was performed with two additional cell lines (LNCaP and VCaP) and can theoretically be performed with all cells expressing EpCAM.

1. Preparation of Target Cells

1. Cell culture and labeling of cells
   NOTE: In this protocol, cells are cultured in 75 cm² culture flasks. Please adjust the amounts of reagents accordingly if other cell culture devices are used (....

Results

Figure 1: Visualizing labeled cells. (A,E) Cells before charging: Cells are labeled with CFSE and counterstained with a DNA intercalating dye showing a range of CFSE (green) signal intensity. (B,F) Labeled cells captured on the wire. (C,G) Wire after cell-detachment .......

Materials

Name	Company	Catalog Number	Comments
Gilupi Release Buffer	Gilupi	GIL083	Used to detach cells from the wire
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
Foetal 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
Vacutainer EDTA (or Na-heparin) tubes	BD	366450 (or 366480)	Used as reaction tube for ex vivo capture of target cells
Detektor CANCER03 DC03	Gilupi	GIL003	Functionalized wire capable of isolating and detaching EpCAM-positive 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
Sterile syringe filter (0.2 µm)	Corning	431219	For filtering freshly prepared Gilupi Release Buffer
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
Axiovert M200 equipped with Mikromanipulator MMJ and CellTram vario	Zeiss/Eppendorf		Use micromanipulation at hand
Microcapillaries (25 µm in diameter), CustomTip Type I	Eppendorf	930001201	Used for micromanipulating cells
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
MembraneSlide 1.0 PET	Zeiss	415101-4401-050	Membrane-coated glass slides used for laser microdissection