( ...)Flow cytometric analysis of human,,,primary cells using the Agilent 2100,,,bioanalyzer and on-chip staining

A fast and accurate method to detect green fluorescent protein expression in samples of low cell numbers
This Application Note addresses the major problems that many researchers working with primary cells have, for example, limited availability, quantity and lifespan. Using the Agilent 2100 bioanalyzer and Cell Fluorescence LabChip kit the transfection efficiency of primary cells can be determined using green fluorescent protein (GFP) as a reporter molecule. Two types of cells were transfected and analyzed: human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts (NHDF). 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 easy-of-use.
Materials and Methods
Primary cell culture
HUVEC, NHDF, culture media, and trypsin/EDTA solution were obtained from Clonetics. HUVEC cells were maintained in EGM-2 medium and NHDF cells were cultured in FGM-2 medium.
Transfection
Cells were trypsinized and seeded into 12-well culture plates at a density of 3 105 cells/mL in 1 mL of culture medium and incubated for 20 hours under cell culture conditions. Prior to transfection the growth medium was replaced by 1 mL of OPTI-MEM I (Gibco). DNA-Lipofectamine 2000 (Invitrogen) complex was prepared and added to the cells according to the supplier's protocol. After six hours of incubation, the transfection medium was removed and replaced with 1 mL of culture medium. The cells were harvested 18 hours later.
On-chip CBNF staining and steps
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