This article describes a method for flow cytometric analysis of human primary cells using the Agilent 2100 bioanalyzer and on-chip staining. The transfection efficiency of primary cells can be determined using green fluorescent protein (GFP) as a reporter molecule. The advantages of using the compact 2100 bioanalyzer for monitoring transfection efficiency in primary cells include low cell consumption, high reproducibility of results, fast on-chip staining procedure, and ease of use.
This document details an application protocol for determining transfection efficiency in human primary cells, specifically Human Umbilical Vein Endothelial Cells (HUVEC) and Normal Human Dermal Fibroblasts (NHDF), using the Agilent 2100 bioanalyzer. The method utilizes a concentrated cell buffer within the Cell Fluorescence LabChip kit to handle low cell numbers effectively.
The protocol involves on-chip staining with the live cell stain CBNF and analysis of green fluorescent protein (GFP) expression as a reporter molecule. Results demonstrate that this approach requires minimal sample consumption, offers high reproducibility, and provides data quality comparable to conventional flow cytometry.
Only 30,000 cells were required for on-chip staining of primary cells and subsequent measurement.
The cells are stained on-chip with the live cell stain CBNF.
A ratio of 1:6 was found to be optimal to obtain a maximum number of live transfected cells.