A protocol to effectively create single cell suspensions of adherent cells for multiparameter high-throughput flow...

Area of Science:

• Cell Biology
• Biotechnology
• Cancer Research

Background:

• High-throughput flow cytometry is challenging for adherent cells due to dissociation-induced cell damage and artificial results.
• Standard trypsinization methods yield low single cell suspension rates, limiting analysis.
• Investigating cellular responses in adherent cancer cell lines requires robust dissociation techniques.

Purpose of the Study:

• To develop and validate an improved protocol for dissociating adherent human cells for high-throughput flow cytometry.
• To enhance single cell suspension yield without compromising cell viability or experimental outcomes.
• To enable accurate analysis of drug-induced cellular alterations in adherent cancer models.

Main Methods:

• Adherent human pancreatic carcinoma cell lines (Panc-1, AsPC-1) were treated with doxorubicin.
• Cells were dissociated using a novel collagenase-trypsin mixture in 96-well plates.
• Fluorescence-activated high-throughput flow cytometry assessed cell viability, cell cycle (7-aminoactinomycin D), and intracellular protein expression (activated caspase-3).

Main Results:

• The collagenase-trypsin protocol increased single cell events from 31.9% (trypsin alone) to 62.1%-85.5%.
• Cell viability remained unaffected by the enhanced dissociation method.
• Doxorubicin-induced protein expression, including activated caspase-3, was accurately detected without interference.

Conclusions:

• The collagenase-trypsin protocol offers a significant improvement in single cell suspension yield for adherent cells.
• This method is compatible with high-throughput flow cytometry and preserves drug-induced cellular signaling.
• The protocol facilitates efficient and reliable investigation of cellular pathways in adherent human cell lines.