Single Cell Sampling Using Micromanipulation: A Technique to Isolate Single Cells from Cancer Cell Suspension

Protocollo

1. Single-cell Sampling

1. 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.
   1. 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.      
   2. 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).
   3. Pipette the entire target cell suspension onto the glass slide.
   4. Transfer the glass slide to a microscope equipped with a micromanipulator. Let the cells settle for 5 min.
   5. 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.
   6. 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.
   7. 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.

Materiali

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