The use of HaloTag-based technology in flow and laser scanning cytometry analysis of live and fixed cells
© Barteneva et al; licensee BioMed Central Ltd. 2011
Received: 11 June 2011
Accepted: 9 September 2011
Published: 9 September 2011
Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.
Here, 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.
Use 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.
One limitation of the fluorescent proteins commonly used in the generation of fusion proteins is that new constructs must be created if different colors are required for analysis. In addition to the labor involved, changing the fusion partner can result in dysregulated localization and/or affect the activity of the protein being analyzed . Another problem is that fluorescent proteins are often prone to fixation, making difficult to combine flow cytometric analysis with cell cycle studies or research of infection agents like HIV or mycobacteria which requiring high percentage of fixation and long fixative time. An alternative approach is to use a fusion partner like HaloTag, SNAP-Tag, FIAsH, or others [2–5] that can be "tagged" later with an exogenous fluorescent ligand. Labeling of these protein tags with small synthetic ligands depends on the formation of stable complexes between biarsenical compounds and peptides containing a tetracysteine thiol motif , and is adaptable to a wide range of cell types . The "tag" technologies have been utilized in diverse experimental procedures, but mainly for in vitro and in vivo imaging of cells with microscopy and immunocytochemistry [4, 7, 8].
Here we describe the successful application of flow cytometry and laser scanning cytometry (LSC) to cells expressing HT constructs. The HT protein is an engineered monomeric haloalcane dehalogenase from Rhodococcus rhodochrous capable of covalent binding to ligands of interest. The HT ligands harbor reactive linkers that covalently bind to the HT protein and fluorescent reporter groups or affinity handles such as biotin, Oregon Green, tetramethylrhodamine (TMR) and others.
We illustrate the utility of this approach with several different systems: i) surface staining of a U2OS human osteosarcoma cell line that is stably transfected with a vector encoding β1-integrin-HaloTag7 (U2OS-β1Int-HT7); ii) intracellular staining of Jurkat CD4 T cells infected with an HIV-1 reporter virus (HIV-1Lai-Halo) that encodes an unfused HT; and iii) intracellular staining of a HEK-293 cell line that expresses a HT fusion with the nuclear factor (NFκB) p65 subunit. The β1Int-HT7 construct is well expressed and tolerated in multiple mammalian cell types .
We also performed intracellular staining of the HEK-293 cell line stably expressing HT-p65 fusion protein. Cells were fixed with 1% PFA for 1 h at 4°C following a 30-min permeabilization at 4°C with 70% ethanol and staining with the Alexa 488 HT ligand (30 min × 37°C w/o fixation for Oregon Green). As shown in Figures 2E and 2F, ligand concentration can be optimized in order to achieve a good signal-noise ratio.
Analysis of HT expression using cytometers equipped with green-yellow lasers (laser excitation 561 nm) and green lasers (534 nm) enables the use of a versatile set of ligands, including TMR (494Ex/516Em) or HT-DiAcFAM ligand (494Ex/526Em). Before introduction of green-yellow and green lasers in the standard cytometer optical configuration, the availability of HT ligands for cytometry applications was limited by HT ligands excited with the 488 nm laser (Oregon Green and Alexa 488) and biotin-conjugated ligands. However, because biotin is an essential co-factor in the cytosol and in mitochondria, the use of biotin-conjugated ligands may result in a significant level of nonspecific binding, especially in intracellular applications.
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. The cytometric approach described herein should be applicable to other "tagged" proteins that utilize a diverse selection of fluorescent ligands (SNAP-tag, etc.) and to a wide range of host cells. In addition, since the HT ligand is connected by a stable covalent bond to its target protein, the fluorescently labeled HT fusion protein can be characterized by SDS-PAGE without loss of fluorescent signal (4).
The combination of HT technology with flow and imaging cytometry analysis has major advantages over microscopy-based methods. As shown here, these cytometric methods allow investigators to statistically assess the expression level of multiple proteins in individual cells within a heterogeneous cell population, and can be combined with cell cycle analysis, viability evaluation, and cell surface/intracellular staining, although nonspecific binding of HT ligands to untagged cellular components remains an issue. Taken together, the newly implemented applications for HT technology described herein should promote the adoption of rapid, efficient and quantitative multiplexing of "tagged" proteins with cell cycle dyes and fluorochrome-conjugated antibodies.
List of abbreviations used
vector encoding β1-integrin-HaloTag7
diacetyl derivative of fluorescein
enhanced green fluorescent protein
fetal bovine serum
fluorescein arsenical hairpin
human immunodeficiency virus
human immunodeficiency virus type 1 (HIV-1) reporter virus expressing the unfused HT
laser scanning cytometry
mean fluorescent intensity
phosphate buffered saline
SDS-polyacrylamide gel electrophoresis
is a 20 kDa mutant of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) that reacts specifically and rapidly with benzylguanine and benzylchlopyrimidine derivatives carrying a variety of different synthetic fluorophores
Funding was provided in part by NIH and the Immune Disease Institute to N.B. and A.G. We are grateful to Dr. Georgiy Los and Natasha Karassina for providing the U2OS cell line expressing the HT-β1 integrin fusion protein and the HEK-293 cell line expressing the HT-p65 fusion protein. We also thank Dr. Elena Holden and Ed Luther for access to the laser scanning cytometric instrumentation.
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