Overview
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
- 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.- 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-(1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol (10% solution), 2.6 µL of a proteinase K solution, and 12.8 µL RNase/DNase-free water to obtain a master mix for 10 samples (total volume 20 µL).
NOTE: For more samples, increase the volumes accordingly. - Use a grease pen to draw an area holding the cell suspension in place. Adjust area and volume if the cell density is too high (i.e., mark a larger area on the glass slide, load 1x PBS and an aliquot of the cell suspension).
- Pipette the entire target cell suspension onto the glass slide.
- Transfer the glass slide to a microscope equipped with a micromanipulator. Let the cells settle for 5 min.
- Prepare 0.2 mL PCR tubes loaded with 2.0 µL of cell lysis master mix (prepared in step 1.1.1.) and store it on ice.
- Collect single cells in 1 µL of 1x PBS using the micromanipulator and transfer the cells into a tube containing cell lysis master mix.
NOTE: For transferring micromanipulated cells into the cell lysis buffer, place the capillary directly into the 2 µL of lysis solution and eject until bubbles can be seen. - Briefly spin down the sample at 2000 x g for 3 s using a desktop microfuge to collect all liquid at the bottom of the tube. Place the samples on ice.
- 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-(1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol (10% solution), 2.6 µL of a proteinase K solution, and 12.8 µL RNase/DNase-free water to obtain a master mix for 10 samples (total volume 20 µL).
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Materials
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 |