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Using Organic Fluorescent Probes in Combination with GFP—Note 12.1

Probes for Multiplexed Detection of GFP-Expressing Cells

The green-fluorescent protein (GFP) reporter has added a new dimension to the analysis of protein localization, allowing real-time examination in living cells of processes that have conventionally been observed through immunocytochemical "snapshots" in fixed specimens. Using spectrally distinct, organic fluorescent probes and markers (Table 1) adds extra data dimensions and reference points to these experiments (Figure 1)

Figure 1. The morphology of sporulating Bacillus subtilis in the early stages of forespore engulfment. The membranes and chromosomes of both the forespore and the larger mother cell are stained with FM 4-64 (red; T3166, T13320) and DAPI (blue; D1306, D3571, D21490), respectively. The small green-fluorescent patch indicates the localization of a GFP fusion to SPoIIIE, a protein essential for translocation of the forespore chromosome that may also regulate membrane fusion events (see Proc Natl Acad Sci U S A 96, 14553 (1999)). The background contains sporangia at various stages in the engulfment process stained with MitoTracker Green FM (green, M7514) and FM 4-64 (red).

The majority of the applications summarized in Table 1 involve living cells, tissues and organisms. There are many other instances where research objectives call for complementary use of immunochemical and GFP-based protein localization techniques. These experiments demand the unmatched combination of brightness, photostability and spectral separation provided by our Alexa Fluor dye–labeled secondary detection reagents. For two-color combinations with GFP, we recommend the Alexa Fluor 555, Alexa Fluor 568 or Alexa Fluor 594 dye–labeled secondary antibodies (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1). The addition of Alexa Fluor 635 or Alexa Fluor 647 dye-labeled antibodies allows three-color detection. Some immunohistochemical procedures such as paraffin embedding of fixed tissue result in loss of the intrinsic fluorescence of GFP. In other cases, GFP expression levels may simply be too low for detection above background autofluorescence. Antibodies to GFP provide remedies for these problems (Figure 2). We offer unlabeled mouse monoclonal and rabbit polyclonal antibodies to GFP (A6455, A11120, A11121, A11122), as well as Alexa Fluor dye–labeled rabbit polyclonal antibodies to GFP (A21311, A21312, A31851, A31852; Primary Antibodies for Diverse Applications—Section 7.5).

Figure 2. HeLa cell transfected with pShooter pCMV/myc/mito/GFP, then fixed and permeabilized. Green-fluorescent protein (GFP) localized in the mitochondria was labeled with mouse IgG2a anti-GFP antibody (A11120) and detected with orange-fluorescent Alexa Fluor 555 goat anti–mouse IgG antibody (A21422), which colocalized with the dim GFP fluorescence. F-actin was labeled with green-fluorescent Alexa Fluor 488 phalloidin (A12379), and the nucleus was stained with blue-fluorescent DAPI (D1306, D3571, D21490). The sample was mounted using ProLong Gold antifade reagent (P36930). Some GFP fluorescence is retained in the mitochondria after fixation (top), but immunolabeling and detection greatly improve visualization (bottom).

Alexa Fluor Dye—Highly Fluorescent FRET Acceptors

Proximity-dependent fluorescence resonance energy transfer (FRET) allows detection of protein–protein interactions with much higher spatial resolution than conventional diffraction-limited microscopy.

Alexa Fluor dyes with strong absorption in the 500–600 nm wavelength range are excellent FRET acceptors from GFP (Table 2). An assay to detect activation of GFP–GTPase fusions developed by researchers at Scripps Research Institute

utilizes the GTPase-binding domain (PBD) of PAK1, a protein that binds to GTPases only in their activated GTP-bound form. GTPase activation is indicated by FRET from GFP to PDB labeled with Alexa Fluor 546 C₅-maleimide at a single N-terminal cysteine residue. This assay has been used to determine the location and dynamics of rac and Cdc42 GTPase activation in live cells.

Normalizing Expression and Translation Signals

In 2002, researchers in Scott Fraser's laboratory at the California Institute of Technology reported a method of coinjecting Texas Red dye–labeled 10,000 MW dextran and GFP vectors into sea urchin embryos. This method overcomes a multitude of problems inherent in making intra- and inter-embryo comparisons of gene expression levels using confocal microscopy. In particular, laser excitation and fluorescence collection efficiencies vary with the depth of the fluorescent protein in the embryo, and the orientation of different embryos on the coverslip varies relative to the microscope objective. Measuring the ratio of GFP fluorescence to Texas Red dextran fluorescence corrects for these spatial factors, providing a gene expression readout that is 2–50 times more accurate than conventional confocal microscopy procedures depending on the localization of GFP within an embryo.

A similar strategy was previously used to determine translation efficiencies of GFP-encoding mRNAs.

Table 1. Probes for multiplexed detection of GFP-expressing cells.*

Target	Probe	Cat #	Ex/Em †	GFP Fusion Partner	Specimen	Reference
Physiological Indicators
Intracellular Ca²⁺	Fura-2 AM	F1201, F1221, F1225, F14185	335/505 ‡	Protein kinase C (PKC)	BHK cells	Biochem J (1999) 337 (Pt 2):211
Intracellular Ca²⁺	X-Rhod-1 AM	X14210	580/602	580/602 Trpm5 (melastatin-related cation channel)	CHO cells	Nat Neurosci (2002) 5:1169
Intracellular Ca²⁺	Fura Red AM	F3020, F3021	488/650	GFP expressed specifically in pancreatic β-cells	Mouse pancreatic islets	Am J Physiol Endocrinol Metab (2003) 284:E177
Intracellular pH	5-(and 6-)Carboxy SNARF-1 AM ester acetate	C1271	568/635	Human growth hormone (hGH)	RIN1046-38 insulinoma cells	Am J Physiol Cell Physiol (2002) 283:C429
Mitochondrial membrane potential	TMRM	T668	555/580	Cytochrome c	MCF-7 human breast carcinoma, HeLa	J Cell Sci (2003) 116:525
Superoxide (O₂^–)	Dihydroethidium	D1168	518/605	Cytochrome c	MCF-7 human breast carcinoma	J Biol Chem (2003) 278:12645
Synaptic activity	FM 4-64	T3166, T13320	506/750 §	VAMP (vesicle-associated membrane protein)	Rat hippocampal neurons	Nat Neurosci (2000) 3:445
Receptors and Endocytosis
Acetylcholine receptor	Tetramethylrhodamine α-bungarotoxin	T1175	553/577	Rapsyn (receptor-aggregating protein)	Zebrafish	J Neurosci (2001) 21:5439
Epidermal growth factor (EGF)	Rhodamine EGF	E3481	555/581	EGF receptor	MTLn3 rat mammary adenocarcinoma	Mol Biol Cell (2000) 11:3873
Endosomes	Transferrin from human serum, Alexa Fluor 546 conjugate	T23364	556/573	β2-adrenergic receptor (β2AR)	HEK 293, rat hippocampal neurons	Brain Res (2003) 984:21
Endosomes	Transferrin from human serum, Alexa Fluor 568 conjugate	T23365	578/603	PrP^c (cellular prion protein)	SN56 cells	J Biol Chem (2002) 277:33311
Endosomes	FM 4-64	T3166, T13320	506/750 §	PrP^c (cellular prion protein)	SN56 cells	J Biol Chem (2002) 277:33311
Organelles
Endoplasmic reticulum	ER-Tracker Blue-White DPX	E12353	375/520 ‡	HSD17B7 gene product (3-ketosteroid reductase)	HeLa, NIH 3T3	Mol Endocrinol (2003) 17:1715
Golgi complex	BODIPY TR ceramide	D7540	589/617	PrP^c (cellular prion protein)	SN56 cells	J Biol Chem (2002) 277:33311
Lysosomes	LysoTracker Red	L7528	577/590	Heparanase	Primary human fibroblasts, MDA-231 (human breast carcinoma)	Exp Cell Res (2002) 281:50
Mitochondria	MitoTracker Red	M7512	578/599	Sam5p (mitochondrial carrier for S-adenosylmethionine)	Yeast (Saccharomyces cerevisiae)	EMBO J (2003) 22:5975
Nuclear DNA	DAPI	D1306, D3571, D21490	358/461	Histone H2B	HeLa	Methods (2003) 29:42
Nuclear DNA	Hoechst 33342	H1399, H3570, H21492	350/461	Histone H1	BALB/c 3T3 fibroblasts	Nature (2000) 408:877
Nuclear DNA	SYTO 17	S7579	621/634	HIV-1 integrase	HeLa	J Biol Chem (2003) 278:33528
Nuclear DNA	SYTO 59	S11341	622/645	Microtubule plus-end binding protein	Porcine kidney epithelial cells (LLCPK)	Mol Biol Cell (2003) 14:916
Nuclear DNA	TO-PRO-3	T3605	642/661	Citron kinase	HeLa	J Cell Sci (2001) 114:3273
Plasma membrane	DiI	D282, D3911, N22880	549/565	Synaptobrevin	Xenopus optic neurons	Nat Neurosci (2001) 4:1093
Other Subcellular Structures
F-actin	Rhodamine phalloidin	R415	554/573	ERM (ezrin-radixin-moesin) proteins	Human peripheral blood T cells (PBT)	Nat Immunol (2004) 5:272
F-actin	Alexa Fluor 568 phalloidin	A12380	578/603	Calponin	NIH 3T3	J Cell Sci (2000) 113 (Pt 21):3725
Lipid rafts	Cholera toxin subunit B (recombinant), Alexa Fluor 594 conjugate	C22842	590/617	Histocompatibility leukocyte antigen (HLA)-Cw4	NK cell–B-cell immunological synapse	Proc Natl Acad Sci U S A (2001) 98:14547
Cell Classification Markers
Apoptotic cells	Annexin V, Alexa Fluor 594 conjugate	A13203	590/617	GRASP65 (Golgi stacking protein)	HeLa	J Cell Biol (2002) 156:495
Transformed B lymphocytes (Raji cells)	CellTracker Orange CMTMR	C2927	550/575	ICAM-3 (intercellular adhesion molecule-3)	T-lymphocytes and antigen-presenting B cells	Nat Immunol (2002) 3:159
Cell-surface antigens	R-Phycoerythrin (streptavidin conjugate)	S866, S21388	565/575	GFP gene expression	NIH 3T3	Cytometry (1996) 25:211
Neurons	NeuroTrace 530/615 red-fluorescent Nissl stain	N21482	530/620	Tau microtubule-binding protein (Purkinje cell marker)	Mouse brain slice	J Neurosci (2003) 23:6392
Neurons	Alexa Fluor 594 hydrazide	A10438, A10442	588/613	Synaptophysin	Aplysia californica sensory neurons	Neuron (2003) 40:151
* This list covers only Aequoria victoria GFP, optimized mutants (e.g., EGFP) and green-fluorescent proteins from other species (e.g., Renilla reniformis). Fluorescent proteins with distinctly different excitation and emission characteristics (CFP, YFP, dsRed, etc.) are not included. † Fluorescence excitation (Ex) and emission (Em) maxima, in nm. ‡ Simultaneous imaging of GFP with fura-2 or ER-Tracker Blue-White DPX requires excitation wavelength–switching capability, because the fluorescence emission spectra overlap extensively. Even under these conditions, signal bleedthrough from one detection channel to the other may still be problematic, depending on the expression level and localization of the GFP chimera. See Biochem J 356, 345 (2001) for further discussion. § The fluorescence emission spectra of styryl dyes such as FM 1-43 and FM 4-64 are broad and extend into the green emission range of GFP. In some cases, FM dye emission can overspill into the GFP detection channel, causing degraded resolution of image features. The excitation and emission spectra of FM 1-43 overlap those of GFP more extensively than those of FM 4-64. Therefore, using FM 4-64 instead of FM 1-43 is recommended to minimize this problem.

Table 2. R₀ values for FRET from EGFP to Alexa Fluor dyes. *

Acceptor Dye	R₀ (Å)
Alexa Fluor 546 dye	57
Alexa Fluor 555 dye	63
Alexa Fluor 568 dye	54
Alexa Fluor 594 dye	53
* R₀ values in angstroms (Å) represent the distance at which fluorescence resonance energy transfer from the donor dye to the acceptor dye is 50% efficient. Values were calculated from spectroscopic data as outlined (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2 ).