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[Beckman Coulter] CytoFLEX SRT, Sorting of PI-negative cells from human pancreatic cancer cell lines
드림셀인스
2024-08-16
조회수 339
Proteome alterations during clonal isolation of established human pancreatic cancer cell lines
- PMID: 36271971
- PMCID: PMC9587952
- DOI: 10.1007/s00018-022-04584-9
Keywords: FACS; Limiting dilution; Mass spectrometry; Single-cell Isolation
Assay: Sorting of PI-negative cells of human pancreatic cancer cell lines, Research Area: Human pancreatic cancer cell linesSpecies: Human
Sample Type: Pancreatic cancer cell linesAdditional Notes: CytoFlex SRT cell sorter operated at 15 psi with a 100 μm nozzle.
Summary
Clonal isolation is an integral step of numerous workflows in genome editing and cell engineering. It comprises the isolation
of a single progenitor cell from a defined cell line population with subsequent expansion to obtain a monoclonal cell
population. This process is associated with transient loss of cell–cell contacts and absence of a multicellular microenvironment.
Previous studies have revealed transcriptomic changes upon clonal isolation with cell line specific extent. Since
transcriptome alterations are only partially reflected on the proteome level, we sought to investigate the impact of clonal
isolation on the cellular proteome to a depth of > 6000 proteins in three established pancreatic cancer cell lines. We show
that clonal isolation does have an impact on the cellular proteome, however, with cell line specific extent, affecting different
biological processes, and also depending on the isolation method. We demonstrate a different impact of clonal isolation on
mesenchymal- and epithelial-derived cell lines mainly affecting cell proliferation, metabolism, cell adhesion and cellular
stress. The results bear relevance to the field of genomic editing and cell engineering and highlight the need to consider the
impact of clonal isolation when interpreting data stemming from experiments that include this step.
Flow cytometry
Clonal isolation by limiting dilution cloning or FACS
For single-cell sorting via FACS, cells were detached and counted like described above before adjusting cell number to 3x 107 cells/mL in the respective medium supplemented with a final concentration of 1 µg/mL propidium iodide (PI, Sigma-Aldrich) as viability marker. FACS-assisted single- cell sorting for PI-negative cells into 96-well tissue culture plates was kindly performed by the Lighthouse Core Facility (Center for Translational Cell Research, University Medical Center Freiburg) using a CytoFlex SRT cell sorter (Beckman Coulter) operated at 15 psi with a 100 µm nozzle. Sub- sequently, cells were cultured and inspected like described above.
Future analysis
- Protein extraction and proteomic sample preparation.
- TMTpro‑16plex HEK‑E.Coli Benchmark Dataset.
- Mass spectrometry measurement
- Proteomic data analysis
Single‑cell isolation via FACS introduces different proteome changes
An alternative method to limiting dilution for single-cell isolation is fluorescence-activated cell sorting (FACS). To evaluate if the proteome changes introduced by FACS-assisted single-cell sorting are comparable or different to cells subjected to limiting dilution cloning, an additional experiment was exemplarily performed with MIA PaCa-2 cells. Therefore, reference cells were compared to cells undergoing a single round of clonal isolation but with different methods including limiting dilution or FACS. For FACS-isolated cells, no morphological or morphometric differences could be observed on the basis of phase contrast and fluorescence microscopy images (Sup. Fig. 18). The resulting proteomic dataset revealed the identification and quantification of 11,313-12,361 peptides and 3791-4166 proteins per fraction (Sup. Fig. 19b) leading to a total of 6855 unique proteins with a TMT-labelling efficiency of 99.8%.
Fig. 18 Cell Morphology before and after Clonal Isolation of human pancreatic MIA PaCa-2 cells via two rounds of limiting dilution or via FACS-assisted single-cell isolation.
Conclusion
This study shows that clonal isolation can have an impact on the cellular proteome.
However, the extent and the biological processes that are affected seem to be cell line- and method-specific. In this study, we could show a different impact of clonal isolation on mesenchymal-derived cell lines and epithelial-derived cell lines. While the clonal isolation of the mesenchymal-derived cell lines MIA PaCa-2 and PANC-1 revealed a downregulated metabolism in the presence of upregulated protein representatives for cell stress, the epithelial-derived cell line AsPC-1 showed an upregulated metabolism without significant abundance changes of the respective cell stress proteins.
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Beckman Coulter Life Sciences
CytoFLEX SRT Benchtop Cell Sorter
Optical Filters: 450/45, 525/40 (2), 585/42, 610/20 (3), 660/10, 675/30, 690/50, 710/50, 712/25, 780/60 (3)Lasers: 405 nm, 488 nm, 561 nm, 638 nmNozzle Diameter(s): 100 µm
Sorting Performance: With a target population of 5% and a threshold rate of <10,000 events per second, >99% purity and >80% of Poisson's expected yield
Collection Vessels: 12x75 mm tubes, 15 mL conical tubes, 96-well plates, 96-deep-well plates, 6-, 24-, 48-, 96- and 384-well plates, slides
Optical Filters: 450/45, 525/40 (2), 585/42, 610/20 (3), 660/10, 675/30, 690/50, 710/50, 712/25, 780/60 (3)
Lasers: 405 nm, 488 nm, 561 nm, 638 nm
Nozzle Diameter(s): 100 µm
Sorting Performance: With a target population of 5% and a threshold rate of <10,000 events per second, >99% purity and >80% of Poisson's expected yield
Collection Vessels: 12x75 mm tubes, 15 mL conical tubes, 96-well plates, 96-deep-well plates, 6-, 24-, 48-, 96- and 384-well plates, slides
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