Automated Quantification of Subcellular Particles in Myogenic Progenitors

Area of Science:

• Cell Biology
• Microscopy
• Biochemistry

Background:

• Fluorescence microscopy visualizes protein localization within cells.
• Subcellular particles like mRNP granules and autophagosomes are crucial for cellular homeostasis and waste management.
• Existing methods for quantifying these particles can be subjective and labor-intensive.

Purpose of the Study:

• To develop an automated, non-biased method for detecting and quantifying subcellular particles.
• To apply this method to messenger ribonucleoprotein (mRNP) granules and autophagosomes.
• To provide a detailed protocol for researchers, including cell isolation and microscopy techniques.

Main Methods:

• Utilized immunocytochemistry and fluorescence microscopy.
• Employed the CellProfiler software for automated image analysis.
• Developed a pipeline for quantifying particle numbers from deconvolved Z-stacks and maximum intensity images.
• Included protocols for isolating primary mouse myoblasts.

Main Results:

• Successfully established an automated pipeline for quantifying subcellular particles.
• Demonstrated the application of the method to both mRNP granules and autophagosomes.
• Quantified the number of subcellular particles per cell using the CellProfiler pipeline.

Conclusions:

• The described automated method offers a robust and unbiased approach for subcellular particle quantification.
• This technique is widely applicable for studying particle dynamics in various biological contexts, including homeostasis and disease.
• The protocol facilitates reproducible research in cell biology and related fields.