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

Protocol

1. Single-cell Sampling

1. 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.
   1. 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-polyethylene glycol (10% solution), resp., 2.6 µL of a proteinase K solution and 34.8 µL of RNase/DNase-free water to obtain a master mix for 10 samples (total volume 45 µL). Place the master mix on ice.
      NOTE: For more samples, increase the volumes accordingly.
   2. UV-treat the membrane-coated slides suited for laser microdissection. Place the slides in a DNA crosslinker device and expose them to the highest UV for about 10 min.
   3. Assemble the membrane-coated slide in a cytocentrifuge and cytospin the entire cell suspension at 300 x g for 5 min.
      NOTE: When using an in-liquid system, where the cells are cytocentrifuge onto the slide in solution, remove the supernatant after cytocentrifugation and apply a dry-spin at 1140 x g for 1 min.
   4. Directly proceed to laser microdissection or let the cytospins air-dry overnight and freeze the samples at -20 °C for long-term storage.
   5. Pipette 4.5 µL of cell lysis master mix into the cap of a 0.2 mL PCR tube and harvest single cells by means of laser microdissection.
   6. Retrieve the PCR tube from the laser microdissection microscope and close the tube. Collect all liquid at the bottom of the tube by short-spin and place the sample on ice.
   7. Proceed with single-cell WGA or store the isolated samples at -80 °C for up to 1 month before further processing.

Materials

Name	Company	Catalog Number	Comments
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 equipment	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