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PH-sensitive fluorescent dyes: are they really pH-sensitive in cells?

ABSTRACT: Chemically synthesized near-infrared aza-BODIPY dyes displayed off-on fluorescence at acidic pH (pKa = 6.2-6.6) through the suppression of the photoinduced electron transfer and/or internal charge transfer process. The apparent pKas of the dyes were shifted well above physiological pH in a hydrophobic microenvironment, which led to "turned-on" fluorescence in micelles and liposomes at neutral and basic pH. Bovine serum albumin also activated the fluorescence, though to a much lesser extent. When these small molecular dyes entered cells, instead of being fluorescent only in acidic organelles, the whole cytoplasm exhibited fluorescence, with a signal/background ratio as high as ?10 in no-wash live-cell imaging. The dye 1-labeled cells remained highly fluorescent even after 3 days. Moreover, slight variations of the dye structure resulted in significantly different intracellular fluorescence behaviors, possibly because of their different cellular uptake and intracellular activation capabilities. After the separation of cellular components, the fraction of plasma membrane and endoplasmic reticulum showed the highest fluorescence, further confirming the fluorescence activation by membrane structures. The fluorescence intensity of these dyes at different intracellular pHs (6.80 and 8.00) did not differ significantly, indicating that intracellular pH did not play a critical role. Altogether, we showed here for the first time that the fluorescence of pH-sensitive aza-BODIPY dyes was switched intracellularly not by acidic pH, but by intracellular membranes (and proteins as well). The excellent membrane permeability, ultrahigh fluorescence contrast ratio, persistent fluorescent signal, and minimal biological interference of dye 1 make it an ideal choice for live-cell imaging and in vivo cell tracking. These findings also imply that the intracellular fluorescence properties of pH-sensitive dyes should be carefully examined before they are used as pH indicators.

SUBMITTER: Zhang XX

PROVIDER: S-EPMC3647017 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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pH-sensitive fluorescent dyes: are they really pH-sensitive in cells?

Zhang Xiao-Xiang XX Wang Zhe Z Yue Xuyi X Ma Ying Y Kiesewetter Dale O DO Chen Xiaoyuan X

Molecular pharmaceutics 20130325 5

Chemically synthesized near-infrared aza-BODIPY dyes displayed off-on fluorescence at acidic pH (pKa = 6.2-6.6) through the suppression of the photoinduced electron transfer and/or internal charge transfer process. The apparent pKas of the dyes were shifted well above physiological pH in a hydrophobic microenvironment, which led to "turned-on" fluorescence in micelles and liposomes at neutral and basic pH. Bovine serum albumin also activated the fluorescence, though to a much lesser extent. When ...[more]

PMID: 23464828

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Project description:The simultaneous measurement of gene expression for thousands of genes in a single analysis by the microarray technology allows researchers to describe transcriptomes in various samples of interest. Problems with variation in data quality derived from microarray experiments are well known and might result from poor RNA quality, background problems, or sub optimal signal strength. To assess variation due to the fluorescent dye chosen, three different dye pairs were tested for labelling of cDNA in gene expression analysis experiments on a porcine immune focused oligonucleotide microarray (POM3). This in-house oligonucleotide microarray allowed a direct comparison of background fluorescence, Median signal intensities, numbers of spots detected, and resistance to photobleaching between different dye pairs. We tested Alexa Flour 546/647, Cy3/Cy5 as well as Oyster 550/650 all from Genisphere Inc., Hatfield, PA, USA. Keywords: Comparison of fluorescent dyes Each dye pairs were hybridized on two slides. The same two samples were compared on all the slides used in the present study. The liver sample from a healthy animal was labeled with the high wavelength dyes (Oyster 650/Alexa 647/Cy5) and the liver sample from the sick animal was labeled with the low wavelength dyes (Oyster 550/Alexa 546/Cy3). Each slide was scanned 3 times, immediately after hybridization (first round), after 1 month of storage in darkness and after 24 hours on the bench (12 hours of daylight and 12 hours of artificial light). Total RNA from the infected and control liver samples were extracted using RNeasy midi kit (Qiagen, Denmark) and DNase treated using RNase-Free DNase Set (Qiagen). 3DNATM Array 900 expression array detection kits (Genisphere Inc.) were used for the labeling and cDNA synthesis reaction of the RNA in the present study. Three different pairs of fluorescent dyes Oyster 550/650 (Genisphere Inc.), Alexa 546/647 (Genisphere Inc.), and Cy3/Cy5 (Genisphere Inc.) were used in separate labeling reactions. Labeling was done according to the manufacturers protocol for large-scale cDNA synthesis. For the cDNA synthesis 9.2Âµg total RNA from each liver sample was used and mixed with 20U AmpliQ RTenzyme and 10x first strand buffer (Bie og Berntsen, RÃ¸dovre, Denmark). Hybridization and washing were performed according to the manufacturers instructions (Genisphere) using Corning hybridization chambers. For one cDNA hybridization reaction; 6.5Âµl cDNA from each synthesis, 0.5Âµl Salmon sperm (10Âµg/Âµl) and 13.5Âµl 2 x Formamide-Based Hybridization Buffer (3DNA Array 900, Genisphere) was used. A total hybridization mix of 29Âµl was applied under a 22I x 25 mm LifterSlip (Erie Scientific, Portsmouth, NH, USA) carefully avoiding air bubbles. Slides were incubated at 44oC in a water bath over night. Wash buffer 1 (3DNA Array 900, Genisphere) was preheated to 44oC for the post cDNA hybridization wash. For two 3DNA hybridization reaction mixes; 2.5Âµl of each Capture reagent and 26Âµl SDS-Based hybridization buffer was mixed to a final volume of 52Âµl. 26Âµl 3DNA hybridization mix was applied to each slide, and incubated in darkness in a water bath at 50oC for 4 hours. Wash buffer 1 was preheated to 60oC for the post 3DNA hybridization wash. Slides were scanned on a CCD ArrayWoRxe auto (Applied Precision, Issaquah, WA, USA) using different exposure times (0.3 for 595 nm and 1.2 for 685 nm).

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PH-sensitive fluorescent dyes: are they really pH-sensitive in cells?

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pH-sensitive fluorescent dyes: are they really pH-sensitive in cells?

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