Fluorescence with Triton

Funk et al. have used a low-pressure mercury lamp without filter to liberate inorganic tin ions from thin-layer chromatographically separated organotin compounds these were then reacted with 3-hydroxyflavone to yield blue fluorescent chromatogram zones on a yellow fluorescent background [22]. Quantitative analysis was also possible here (XoK = 405 nm, Xji = 436 nm, monochromatic filter). After treatment of the chromatogram with Triton X-100 (fluorescence amplification by a factor of 5) the detection limits for various organotin compoimds were between 200 and 500 pg (calculated as tin). 

Fig. 2 Increase in the fluorescence intensity of dienestrol as a function of heating time after immersion of the chromatogram in sodium hydroxide solution (c = 1 mol/L) followed by treatment with Triton 100-X and heating to 120°C.

Fluoromax-3 spectrofluorometer (Edison, NJ). The total calcein content of the vesicles was determined after lysis of the membranes with Triton X-100 (1% (v/v)). Calcein retention of vesicles was estimated from the fluorescence before and after the addition of Triton X-100. Prior to calcein retention analysis, the freeze-dried samples were rehydrated at room temperature. 

The fluorescence reading at this point is taken to be the maximal fluorescence (Tmax). It can be difficult to solubilize DNA/liposome aggregates with Triton X-100, but heating the samples to the cloud point of the detergent (about 100°C) (104) followed by vortex mixing helps. 

Figure 18 Comparison of the rate of HPTS [8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine)] release from liposomes (eggPC) modified with NIPPAm-containing copolymers and the phase transition of the copolymer (NIPPAm/MAA/ODA 93 5 2 mol%). The copolymer phase transition was measured as Em increase in light scattering at 480 nm. HPTS release was measured as an increase in HPTS fluorescence following release from hposomes (Aex = 413nm, Aem = 512nm) and is expressed as a fraction of the fluorescence upon hposome soluhilization with Triton X-100. (Adapted from Ref. 117.)...

FIGURE 1 Effect of fatty acids on the photostability (top), acid stability (middle) and fluorescence behavior (bottom) of LHC II — comparison with Triton X-100. 

Fig. 12 Fluorescence emission spectrum of AOT-vesicles produced by centrifugation before black) and after treating with Triton X 100 red) [20]...

Cellular interactions on the developed nanoparticles-enriched coatings were also examined by inverted fluorescence microscopy. After 24 and 72 hours of incubation, the replicate surfaces were harvested and washed thrice with PBS. The cells that attached to the surfaces were fixed with paraformaldehyde (4%) for 10 minutes and permeabilized with Triton X-100 (0.1%) for 5 minutes. The actin filaments of the cytoskeleton were labeled with rhodamine phalloidin for 2 hours at room temperature. The surfaces were then mounted using Vectashield with DAPI and examined by an inverted fluorescence microscope with a magnification of 20X. 

The following Tables 2.1 to 2.3 summarize some examples based exclusively on thermochemical reactions on the sorbent surface which lead to the formation of fluorescent reaction products. The derivatives formed frequently remain stable for weeks [6] and the fluorescence can frequently be intensified and/or be stabilized by treatment with viscous liquids (liquid paraffin, Triton X-100, polyethylene glycol etc.). 

Induced fluorescence (X, > 430 nm, cut off filter) by thermal treatment of the chromatogram, the fluorescence increased by a factor of 2.5 by dipping in a solution of Triton X-100 — chloroform (1+4). Working range 2-50 ng substance per chromatogram zone. Prewashing the layers with methanol-ammonia solution (25%) (50+ 50) increased the precision. 

Detection and resnlt The chromatogram was freed from mobile phase for 5 min in a stream of cold air, immersed twice in the dipping solution for 2 s and then dried for 5 min in a stream of cold air. In order to stabilize and enhance the fluorescence intensity it was then immersed twice for 2 s in a solution of Triton X-100 — chloroform (1+4), with the chromatogram being kept in the dark between and after these dipping processes. 

Fig. 1 Fluorescence scan of a chromatogram track with 1 ng cortisone (1), 100 ng dienestrol (2X 300 ng 17a-ethinyl-l,3,5-estratriene-3,17B-diol (3), 100 ng estrone (4) and 1 ug each of 4-androstene-3,17-dione (5) and 4-cholesten-3-one (6) A. before immersion in Triton X-100, B. after immersion followed by brief drying, C after heating to 120 °C for 10 minutes and D. for a further 20 minutes to increase the fluorescence.

Apoptosis assay. ECRF24 or A2780 cells were seeded on 6-well plates (2 X 105 cells/ well) and grown 24 hours in complete medium before treatment. Compounds 1-3 were freshly dissolved in DMSO, diluted in complete medium and added to the cells at the final concentrations indicated in Table 2. After incubation for 72 h apoptosis was measured by flow cytometric determination of subdiploid cells after DNA extraction and subsequent staining with propidium iodide (PI) as described previously10. Briefly, cells were harvested and subsequently fixed in 70% ethanol at —20°C. After 2 h the cells were re-suspended in DNA extraction buffer (45 mM Na2HP04, 2.5 mM citric acid, and 1% Triton X-100, pH 7.4) for 20 min at 37°C. PI was added to a final concentration of 20 pg/mL and log scale red fluorescence was analyzed on a FACSCalibur (BD Biosciences, NJ, U.S.). 

Fig. 6.15. (A) Structure of the PLA2 probe PENN/SATE. (B) Fluorescence spectra change of the PLA2 probe measured over time. The probe (1 pM) was added to a 3 mM solution of Triton XI00 micelles and incubated with bee venom PLA2. The excitation wavelength is at 440 nm. After 36 min the reaction was complete leading to a 30-fold increase in donor/acceptor ratio.

Howard [27] determined dissolved aluminium in seawater by the micelle-enhanced fluorescence of its lumogallion complex. Several surfactants (to enhance fluorescence and minimise interferences), used for the determination of aluminium at very low concentrations (below 0.5 pg/1) in seawaters, were compared. The surfactants tested in preliminary studies were anionic (sodium lauryl sulfate), non-ionic (Triton X-100, Nonidet P42, NOPCO, and Tergital XD), and cationic (cetyltrimethylammonium bromide). Based on the degree of fluorescence enhancement and ease of use, Triton X-100 was selected for further study. Sample solutions (25 ml) in polyethylene bottles were mixed with acetate buffer (pH 4.7, 2 ml) lumogallion solution (0.02%, 0.3 ml) and 1,10-phenanthroline (1.0 ml to mask interferences from iron). Samples were heated to 80 °C for 1.5 h, cooled, and shaken with neat surfactant (0.15 ml) before fluorescence measurements were made. This procedure had a detection limit at the 0.02 pg/1 level. The method was independent of salinity and could therefore be used for both freshwater and seawater samples. 

Fig. 1. (opposite page) Distribution of FITC-conjugated BSA in various fibroblast cell lines under different fixation/permeabilization regimes. (A-D) Protein distribution in living cells (A) PtKj, (B) CHO, (C) 3T3, and (D) HeLa cells. The protein is excluded from the nuclei of all cells. (E-H) Protein distribution in cells extracted for 10 min with 0.1% Triton X-100 before fixation for 30 min with 3.7% formaldehyde (E) PtKi, (F) CHO, (G) 3T3, and (H) HeLa cells. Nuclear fluorescence is seen in (E) PtKj and (G) 3T3 cells. (I-L) Protein distribution in cells extracted for 10 min with 1% Triton X-100 before fixation for 30 min with 3.7% formaldehyde (I) PtKj, (J) CHO, (K) 3T3, and (L) HeLa cells. No fluorescence is detected in the cells with the exception of some nuclear fluorescence seen in (L) HeLa cells. (M-P) Protein distribution in cells fixed for 30 min with 3.7% paraformaldehyde before permeabilization for 10 min with 0.1% Triton X-100. Fluorescence is seen primarily in the cytoplasm with the exception that nuclear fluorescence is seen in (M) PtKi and (N) CHO cells. (Q-T) Protein distributions in cells fixed for 5 min with 90% methanol, 50 vaM EGTA at -20°C (Q) PtKj, (R) CHO, (S) 3T3, and (T) HeLa cells. All cells show an overall low fluorescence, fibrous-textured cytoplasmic fluorescence, and bright staining at the periphery of the nucleus. 10 mm per scale division (black bar). (Reproduced with permission from ref. 6.)... 

Fluorescence of the CF-containing vesicles was measured with an Amino-Bowan spectrofluorometer (excitation 490, emission 550) using a Coming cut-off filter ( 3-68, 520 nm). Complete release was obtained by adding Triton X-100 ( 0.05% v/v). Light scattering was measured with the same instrument with excitation and emission both set to 400 nm. PS concentrations were assayed by phosphate determination (46). 

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