Abstract
BackgroundCombining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.FindingsHere, we describe development of applications using protein tag technology (HaloTag (HT)-based) for flow and laser scanning cytometry (LSC). Cell lines, expressing recombinant surface β1-integrin-HT and HT-p65 fusion protein, and a CD4 T cell line (Jurkat) infected with human immunodeficiency virus type 1 (HIV-1) reporter virus expressing the unfused HT (HIV-1Lai-Halo), were stained with different HT ligands and successfully detected by flow cytometers equipped with 488 and 561 nm lasers as well as a laser scanning cytometer (equipped with 488 and 405 nm lasers) alone or combined with cell cycle and viability markers.ConclusionsUse of HT technology for cytometric applications has advantages over its use in microscopy as it allows for the statistical measurement of protein expression levels in individual cells within a heterogeneous cell population in combination with cell cycle analysis. Another advantage is the ability of the HaloTag to withstand long fixation and high concentration of fixative, which can be useful in research of infectious agents like HIV and/or mycobacteria.
Highlights
Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells
We describe the successful application of flow cytometry and laser scanning cytometry (LSC) to cells expressing HT constructs
The use of interchangeable ligands that recognize the HT and emit in green and red ranges makes it possible to modify a particular experiment in order to (1) avoid undesirable spectral overlapping; (2) decrease autofluorescence with TMR or other red-emitting HT ligands; (3) manipulate colors interchangeably for surface or intracellular staining without the need to generate additional plasmids encoding new fusion partner combinations; and (4) perform extensive cell fixation if infectious agents that require inactivation like HIV or Mycobacterium tuberculosis are being studied
Summary
Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells. The cells were stained with Oregon Green (0.5 μM) or Alexa 488 (1.0 μM) HT ligands (both - Promega, Madison, WI) for 30 min and carefully washed with warm medium; Hoechst 33342 (5 μg/ml), and propidium iodide (PI) (1 μg/ml) were added for 30 min before analysis.
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