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 (receptoraggregating

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

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 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

* 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.