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 live cells of processes that have conventionally been observed through immunocytochemical "snapshots" in fixed specimens.ref Using spectrally distinct, organic fluorescent probes and markers (Table 1) adds extra data dimensions and reference points to these experiments (Figure 1).

Bacillus subtilis   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 (1999) 96:14553). 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 live 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 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.ref Antibodies to GFP provide remedies for these problems (Figure 2). We offer unlabeled mouse and rabbit monoclonal GFP antibodies and unlabeled rabbit and chicken polyclonal GFP antibodies, as well as Alexa Fluor dye–labeled rabbit polyclonal GFP antibodies (Antibodies against Expression Tags—Section 7.5).

pShooter pCMV/myc/mito/GFP
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 (left), but immunolabeling and detection greatly improve visualization (right).

Alexa Fluor Dyes: 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.ref 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 ref 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.ref

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

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

Target Probe Cat. No. 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) 337:211
Intracellular Ca2+ X-Rhod-1 AM X14210 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 PrPc (cellular prion protein) SN56 cells J Biol Chem (2002) 277:33311
Endosomes FM 4-64 T3166, T13320 506/750 § PrPc (cellular prion protein) SN56 cells J Biol Chem (2002) 277:33311
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 PrPc (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) 113:3725
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 (2001) 356:345 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 ).