Overview
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-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. - 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.
- 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. - Directly proceed to laser microdissection or let the cytospins air-dry overnight and freeze the samples at -20 °C for long-term storage.
- 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.
- 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.
- Proceed with single-cell WGA or store the isolated samples at -80 °C for up to 1 month before further processing.
- 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.
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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 |
