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Immunology JoVE (Open Access) Citable · DOI

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

DOI: 10.3791/56394-v
What you'll learn
  • ✓ Enrich rare target cells from mixed populations using functionalized wire technology
  • ✓ Perform single-cell sampling via micromanipulation and laser microdissection
  • ✓ Apply whole genome amplification for downstream single-cell molecular analysis
Protocol

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Difficulty
advanced
Total time
~4–6 hours per sample (including cell preparation, enrichment, sampling, and WGA)

Steps

1
Prepare target cells for enrichment

Process and prepare the cell mixture containing target and background cells for loading onto the functionalized wire system.

▶ 01:28
2
Charge the capture wire

Apply electrostatic or chemical charge to the functionalized wire to enable target cell binding.

▶ 02:41
3
Count and visualize captured cells

Enumerate target cells bound to the wire and verify capture efficiency using microscopy.

▶ 03:27
4
Detach and recover enriched target cells

Release captured cells from the wire and collect them for downstream single-cell processing.

▶ 04:03
5
Sample single cells via micromanipulation

Isolate individual target cells using micropipette-based micromanipulation under microscopic guidance.

▶ 05:25
6
Sample single cells via laser microdissection

Isolate individual target cells using laser-based microdissection and catapulting technology.

▶ 06:37
7
Perform whole genome amplification

Amplify genomic DNA from single cells to sufficient quantity for downstream molecular characterization and analysis.

▶ 07:47
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