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Cell Preparation and Isolation Experimental Design

Cell preparation and isolation experimental design provides physical separation of cells from suspensions and tissues for in-depth analysis by laser capture microdissection, fluorescence-activated cell sorting (FACS), and piezoacoustic sorting with cellenONE. 

Access the workflow steps for each specific cell preparation and isolation experimental design process.

Laser capture microdissection is used to excise cells or cell subsets from sample tissues. The resulting dissections can be analyzed by multi-omic applications downstream. 

The first image shows stomata cells before laser capture microdissection. The second image shows stomata cells after laser microdissection. The third image shows collected tissue in a nanoPOTS chip.
​ ​Laser capture microdissection (LCM) was used to target leaf stomata. The circles in the first two images are the targets that were dissected and collected. Leaf stomata (guard cells) were collected from the abaxial layer of sugarcane leaves. The images are before LCM isolation (left), after LCM collection (middle), and enriched stomata in a Nanodroplet Processing in One Pot for Trace Samples well (right).   (Image provided by William Chrisler | Pacific Northwest National Laboratory)
1. Sample Preparation 
  • Isolate spatially resolved samples from multicellular tissues or biofilms 
  • Cryosection or create a micrograph 
  • Fix sample cross-sections with ethanol and then dehydrate 
2. Cryotomy 
  • Embed sample in an appropriate material (optimal cutting temperature, hydroxypropyl-methyl cellulose polyvinylpyrrolidone, etc.) 
  • Prepare thin sections with a cryostat 
  • Transfer thin section(s) to a polyethylene naphthalate membrane slide 
3. Laser-Microscope Dissection 

  • Scan slide and align with collection device 

  • Image for target identification 

  • Dissect subsections and collect voxels 

  • RNA – short collection  

  • Protein – longer collection 

  • Collect images 

4. Further Analyses 
Contact

Will Chrisler, william.chrisler@pnnl.gov

Fluorescence-Activated Cell Sorting (FACS) is used to characterize cell populations directly or prepare sample subsets for downstream analysis. 

Fluorescence-Activated Cell Sorter (FACS)
The Environmental Molecular Sciences Laboratory's The Fluorescence-Activated Cell Sorter (FACS) allows the collection and enrichment of unique populations of cells, or single cells, from a heterogenous culture.
1. Sample Preparation 
  • Determine if live or fixed cells are required 
  • Apply stain or dye: 
    • immunostaining 
    • vitality stains 
    • organelle stains 
    • fluorescent proteins 
    • activity-based probes 
  • Dilute and declump to generate a single-cell suspension 
  • Remove cell wall (if required) 
2. Initial Cell Population Analysis 
  • Analyze population by cell size/complexity 
  • Sort subset on gate criteria 
  • Verify expectation/accuracy of sorting by confocal microscopy 
3. Sorting 
  • Isolate single cells into 96/384-well formats 
  • Collect bulk subpopulation(s) 
  • Quantify abundance or proportions as an endpoint 
4. Further Analyses 
Contact

Will Chrisler, william.chrisler@pnnl.gov

cellenONE is high-precision single-cell isolation and dispensing system that enables the accurate isolation of individual cells from heterogeneous samples for further analysis. The system operates at a lower pressure relative to that for FACS for handling delicate samples. The workflow involves the precise sorting of single cells and controlled liquid handling down to a picoliter size, all with piezoelectric dispensing for very small volumes. cellenONE is a robust tool for applications in genomics, transcriptomics, and proteomics to aid our understanding of cell heterogeneity and complex biological processes. 

cells deposited in nanowells by CellenONE
Cell sorting technology such as celleONE can isolate single cells of Yarrowia lipolytica  for molecular analysis (proteomics, metabolomics, transcriptomics) after being deposited in nanowells. (Image provided by Erin Bredeweg | Environmental Molecular Sciences Laboratory)
1. Sample Preparation 
  • Determine if live or fixed cells are required 
  • Apply stains or dyes: 
  • Dilute and declump to generate a single-cell suspension 
  • Remove cell wall (if necessary) 
2. Quality Check 
  • Analyze population by cell size/signal 
  • Sort a subset on gate criteria 
  • Collect images (of each sorted cell or item) 
3. Sorting and picoliter dispensing Applications 
  • Sorting single cell into a 96- or 384-well format on different type of modified glass slides like nanoPOTS chip or indium tin oxide-coated glass slide.
  • Dispensing picoliter of samples or reagents onto amino functional glass slide or Cryo-EM Grids.
4. FURTHER ANALYSES
  • mass spectrometry
  • transcriptomics
  • Nanodroplet Processing in One Pot for Trace Samples (nanoPOTS)
  • Nanoscale secondary ion mass spectrometry 
  • Fluorescent in situ hybridization
Contact

Lye Meng Markillie, meng.markillie@pnnl.gov