A Sample Preparation Protocol for High Throughput Immunofluorescence of Suspension Cells on an Adherent Surface

Area of Science:

• Cell Biology
• Proteomics
• Microscopy

Background:

• Imaging techniques like immunofluorescence and confocal microscopy provide crucial in situ spatial information on protein expression at the single-cell level.
• Analyzing non-adherent hematopoietic cells is challenging due to poor surface attachment, leading to cell loss and hindering proteomic profiling.
• Systematic profiling of hematopoietic cells is essential for comprehensive human cell mapping efforts.

Purpose of the Study:

• To develop and establish a robust immunofluorescence protocol for preparing and imaging suspension cells, including cell lines, peripheral blood mononucleated cells (PBMC), and human platelets.
• To enable high-throughput imaging applications for non-adherent cells, overcoming previous technical challenges.
• To facilitate the systematic exploration of protein localization within subcellular structures of hematopoietic cells.

Main Methods:

• Development of an immunofluorescence protocol tailored for suspension cell lines, PBMCs, and platelets.
• Adaptation of the protocol to a multi-well plate format for automated sample preparation.
• Utilization of confocal microscopy for detailed subcellular protein localization analysis.

Main Results:

• Successful establishment of an immunofluorescence protocol for preparing and imaging non-adherent hematopoietic cells on an adherent surface.
• Demonstration of automated sample preparation in a multi-well plate format, enabling high-throughput applications.
• Validation of the protocol for systematic exploration of protein localization to major subcellular structures using confocal microscopy.

Conclusions:

• The developed immunofluorescence protocol effectively overcomes the challenges associated with imaging suspension hematopoietic cells.
• This high-throughput method allows for robust and systematic profiling of protein localization in critical cell types.
• The protocol significantly advances cell mapping efforts by enabling detailed analysis of the hematopoietic proteome.