Isolation of viable type II alveolar epithelial cells by flow cytometry

We developed a new method for isolating viable type II cells from fractionated and unfractionated lung cell suspensions by flow cytometry using acridine orange (AO). Fischer-344 rat lungs were dispersed into single-cell suspensions by a technique that yields a high number of cells (4-5 X 10(8) cells/lung, congruent to 85% viable), congruent to 11% of which are type II cells. Elutriated fractions from the lung cell preparation and parent, unfractionated cell suspensions were incubated with 1.0-0.02 micrograms/ml AO and analyzed by flow cytometry. Parameters analyzed included axial light loss (ALL) and red fluorescence (RF). Based on their unique RF, attributable to AO staining of type II cell lamellar bodies, and their ALL characteristics, type II pneumocytes were sorted from elutriated fractions to greater than 95% purity. Using the same approach, type II pneumocytes were sorted from unfractionated lung cell suspensions at greater than or equal to 85% purity. The viabilities of the type II alveolar epithelial cells isolated by this method range from 85% to 95%, and the ultrastructural features of the sorted cells were unaltered by AO labeling or sorting.     

Plasmodium-infected blood cells analyzed and sorted by flow fluorimetry with the deoxyribonucleic acid binding dye 33258 Hoechst.

Red cells from Plasmodium berghei infected mouse blood can be sorted on the basis of their DNA content with the bisbenzimidazole dye 33258 Hoechst. The optimal conditions for dye uptake have been established and with these conditions uninfected cells are nonfluorescent and can be completely separated from infected cells which exhibit fluorescence in almost direct proportion to the number of parasite nuclei (i.e. DNA) they contain. The number of fluorescent cells detected and their fluorescence intensity is shown to be dependent on the dye concentration and the incubation medium being used. At least a proportion of the infected cells sorted from each fluorescence peak in the cell distribution retain their infectivity in vivo with some, but not all, conditions of labeling. This technique is being used to separate minor cell populations from infected blood for biochemical and immunochemical analyses and to screen human samples for malaria infected cells.     

Automatic cell identification and enrichment in lung cancer: V. Adenocarcinoma and large cell undifferentiated carcinoma

The aims of this study were to develop a protocol for the identification and enrichment of cancer cells from sputum obtained from patients with adenocarcinoma of the lung (n = 6) and large-cell undifferentiated carcinoma of the lung (n = 2), and to compare these findings with the results from our previous studies on other cell types from lung cancer. The hypotheses tested were: Cancer cells in sputum can be preserved following flow sorting. Enrichment for cancer cells from acridine orange (AO)-stained specimens can be achieved. Discrimination of cancer cells from noncancer cells is by AO green fluorescence and discrimination of lymphocytes from other cell types is by AO red fluorescence. Cancer cells are consistently enriched in the AO high green and red fluorescence region, although, for a given cell type, maximal enrichment is patient-dependent. Finally, cancer cell enrichment and lymphocyte exclusion can be done simultaneously. Cells from sputum were initially fixed, stained with AO, sorted on a dual parameter flow sorter, and classified into six groups corresponding to two ranges of green and three ranges of red fluorescence intensities. Cells of each region were stained by the method of Papanicolaou and differential counts were performed to determine the relative frequencies (i.e., purities) of leukocytes, macrophages, squamous cells, and cancer cells, in sorted and unsorted (i.e., control) samples. The average purity of leukocytes (81%), macrophages (6%), squamous cells (11%), and cancer cells (2%) varied markedly from sample to sample. However, the largest enrichment values (i.e., ratio of purity of a cell type in a sorted sample to its purity in the unsorted control sample) achieved for cancer cells consistently occurred for each patient sample in the region corresponding to high green and high red fluorescence intensities. Experimentally, a cancer cell average enrichment of sixteen-fold was obtained by this method. Additionally, fluorescence intensity ranges which increased the enrichment for macrophages by cell sorting typically excluded leukocytes and squamous cells, and vice versa. Finally, red fluorescence intensity was the primary discriminatory parameter for all cell types studied, although the additional use of green fluorescence intensity significantly increased cancer cell enrichment rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Babesia rodhaini, Babesia bovis, and Babesia bigemina: analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst.

The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments.     

Analysis of lateral redistribution of a plasma membrane glycoprotein- monoclonal antibody complex [corrected] [published erratum appears in J Cell Biol 1988 Apr;106(4):following 1403]

The lateral redistribution of a major murine glycoprotein, GP80, was studied on locomoting fibroblasts, using rhodamine-conjugated mAbs and ultralow light level digitized fluorescence microscopy. Confirming an earlier study (Jacobson, K., D. O'Dell, B. Holifield, T.L. Murphy, and J. T. August. 1984. J. Cell Biol. 99:1613-1623), the distribution of GP80 was coupled with cell locomotion; motile cells exhibited a gradated distribution of the GP80-mAb complex over the cell surface, increasing from the front to the rear, whereas stationary cells exhibited a nearly uniform GP80 distribution. By monitoring locomoting single cells, we found the gradated fluorescence distribution to be maintained as an approximate steady state. Newly extended leading edges were almost devoid of the fluorescence labeling. This was strikingly demonstrated in prechilled cells in which the extension of fluorescence-free leading edges caused a pronounced boundary between fluorescent and nonfluorescent zones. Subsequently this boundary eroded gradually in a manner consistent with diffusional relaxation. Evidence indicated that the GP80 redistribution was primarily caused by the lateral motion of GP80 in the plasma membrane and not via intracellular membrane traffic. Two cell locomotion models which, in principle, could account for the GP80 redistribution were tested: the retrograde lipid flow (RLF) model (Bretscher, M. S., 1984. Science (Wash. DC). 224:681-686) and an alternative hypothesis, the retraction-induced spreading (RIS) model. The predictions of these models were stimulated by computer and compared with experiment to assess which model was more appropriate. Whereas both models predicted steady-state gradients similar to the experimental result, only the RIS model predicted the lack of retrograde movement of the fluorescent boundary.     

High zone-selectivity of cell permeabilization following digitonin-pulse perfusion of rat liver. A re-interpretation of the microcirculatory zones

The plasma membrane permeabilization obtained by exposure of hepatocytes to digitonin is utilized in the so-called digitonin-pulse perfusion of rat liver (Quistorff and Grunnet 1987). Brief pulses of digitonin applied with antegrade and retrograde perfusion of the liver caused selective elution of cytosolic enzymes and metabolites from the periportal and the perivenous zone of the same liver. In the present study a light microscopical examination of the liver fixed immediately after the digitonin pulse confirmed the very high zonal selectivity of the method inferred from the marker enzyme pattern of the eluates: Only cells around the port of entry of digitonin were affected and the borderline between affected and non-affected cells was always sharp. The typical periportal lesion was triangular in shape, enclosing the portal space, while the perivenous lesion was roughly circular, concentric with the hepatic vein. Assuming that the digitonin lesion reflects the microcirculatory flow pattern these findings seem to be at variance with the acinar model of Rappaport (Rappaport et al. 1954). The lesion in the lobuli near the surface of the liver as reflected by the discoloration pattern observed on the surface was the same as the lesion of deeper lobuli. The conducting vessels of the liver were only insignificantly affected by digitonin. At the cellular level only the sinusoidal luminal surface of the hepatocytes was affected. The cytoplasmic matrix of the cells including glycogen appeared thinned. All cell types of the liver parenchyma seemed to be equally affected by the digitonin treatment.     

High zone-selectivity of cell permeabilization following digitonin-pulse perfusion of rat liver

Summary The plasma membrane permeabilization obtained by exposure of hepatocytes to digitonin is utilized in the so-called digitonin-pulse perfusion of rat liver (Quistorff and Grunnet 1987). Brief pulses of digitonin applied with antegrade and retrograde perfusion of the liver caused selective elution of cytosolic enzymes and metabolites from the periportal and the perivenous zone of the same liver. In the present study a light microscopical examination of the liver fixed immediately after the digitonin pulse confirmed the very high zonal selectivity of the method inferred from the marker enzyme pattern of the eluates: Only cells around the port of entry of digitonin were affected and the borderline between affected and non-affected cells was always sharp. The typical periportal lesion was triangular in shape, enclosing the portal space, while the perivenous lesion was roughly circular, concentric with the hepatic vein. Assuming that the digitonin lesion reflects the microcirculatory flow pattern these findings seem to be at variance with the acinar model of Rappaport (Rappaport et al. 1954). The lesion in the lobuli near the surface of the liver as reflected by the discoloration pattern observed on the surface was the same as the lesion of deeper lobuli. The conducting vessels of the liver were only insignificantly affected by digitonin. At the cellular level only the sinusoidal luminal surface of the hepatocytes was affected. The cytoplasmic matrix of the cells including glycogen appeared thinned. All cell types of the liver parenchyma seemed to be equally affected by the digitonin treatment.     

Flow cytometry and sorting of meiotic prophase cells of female rabbits

We present a new, flow cytometric method by which cells in various stages of the meiotic prophase can be quantitated and sorted in partly enriched fractions. Ovarian cells of 3-16-day-old rabbits were mechanically dispersed and fixed in ethanol and aldehydes. The cell suspension was stained with the DNA fluorochrome mithramycin and analysed and sorted in a FACS IV cell sorter according to the fluorescence and forward light scatter distribution. Cells sorted onto slides were stained with haematoxylin and eosin and differentially counted in the microscope. In the diploid fraction, preleptotene cells were more fluorescent than somatic cells. Leptotene cells were found throughout the S fraction and the tetraploid fraction. Zygotene and pachytene cells caused a major peak in the tetraploid region with 10-25% more fluorescence than somatic cells. Cells in diplotene had 5-15% more fluorescence than somatic cells. Mitotic cells were 20-40% more fluorescent than somatic cells and scattered the light more intensely than did meiotic cells with the same fluorescence.     

Isolation of anucleate cells using a fluorescence activated cell sorter (FACS II)

Human fibroblasts or mouse teratocarcinoma cells were enucleated by density gradient centrifugation in the presence of cytochalasin B (CB). The resulting mixed population of nucleated and anucleate cells was further purified by flow sorting, using the dye Hoechst 33342 as a fluorescent label for the nucleated cells. The purity of the anucleate cells obtained with this technique was at least 99%, as was shown by histological staining of the sorted fractions. Sorted enucleated fibroblasts were shown to have an intact cell membrane as indicated by their ability to convert fluorescein diacetate into fluorescein and to accumulate this product. They were found to attach and spread when cultured and showed protein synthesis immediately after enucleation, evidenced by the incorporation of [³H]leucine. Sorted enucleated teratocarcinoma cells also had an intact cell membrane, but they did not attach when cultured.     

The metabolism of fructose in the bivascularly perfused rat liver.

The metabolism of fructose was investigated in the bivascularly and hemoglobin-free perfused rat liver. Anterograde and retrograde perfusions were performed. In anterograde perfusion, fructose was infused at identical rates (19 mumols min-1 g-1) via the portal vein (all liver cells) or the hepatic artery (predominantly perivenous cells); in retrograde perfusion fructose was infused via the hepatic vein (all liver cells) or the hepatic artery (only periportal cells). The cellular water spaces accessible via the hepatic artery were measured by means of the multiple-indicator dilution technique. The following results were obtained. (i) Fructose was metabolized to glucose, lactate and pyruvate even when this substrate was infused via the hepatic artery in retrograde perfusion; oxygen consumption was also increased. (ii) When referred to the water spaces accessible to fructose via the hepatic artery in each perfusion mode, the rate of glycolysis was 0.99 +/- 0.14 mumols min-1 ml-1 in the retrograde mode; and, 2.05 +/- 0.19 mumols min-1 ml-1 in the anterograde mode (P = 0.002). (iii) The extra oxygen uptake due to fructose infusion via the hepatic artery was 1.09 +/- 0.16 mumols min-1 ml-1 in the retrograde mode; and, 0.51 +/- 0.08 mumols min-1 ml-1 in the anterograde mode (P = 0.005). (iv) Glucose production from fructose via the hepatic artery was 2.18 +/- 0.18 mumols min-1 ml-1 in the retrograde mode; and, 1.83 +/- 0.16 mumols min-1 ml-1 in the anterograde mode (P = 0.18). (v) Glucose production and extra oxygen uptake due to fructose infusion did not correlate by a single factor in all perfusion modes. It was concluded that: (a) rates of glycolysis are lower in the periportal area, confirming previous views; (b) extra oxygen uptake due to fructose infusion is higher in the periportal area; (c) a predominance of glucose production in the periportal area could not be demonstrated; and (d) extra oxygen uptake due to fructose infusion is not a precise indicator for glucose synthesis.     

An improved resazurin-based cytotoxicity assay for hepatic cells

A simple resazurin-based cytotoxicity assay is presented for screening of cytotoxicity in hepatocytes and liver cell lines. Human hepatoma (HepG2) cells in 96-well culture plates were exposed to known toxic (cisplatin, 5-fluorouracil, ethionine, flufenamic acid, and diflunisal) and control (transplatin, 5-chlorouracil, methionine, and acetylsalicylic acid) compounds for 1–3 days, and resazurin (5 μmol/L) was added. A conventional short-term (1 h) assay was first performed, where cytotoxicity is indicated by decreased reduction of resazurin to its fluorescent product resorufin. Our improved assay consists of additionally measuring fluorescence 2–4 days later, when cytotoxicity is indicated by a striking increase in the concentration of resorufin, resulting from two distinct processes. First, viable liver-derived cells slowly convert resorufin to nonfluorescent metabolites. Fluorescence of control cell wells decreased to background during a 2- to 4-day exposure to resazurin. This metabolism of resorufin was largely blocked by dicumarol and to lesser extents by disulfiram and SKF525a. Second, dead or dying cells slowly convert resazurin to resorufin but do not further metabolize resorufin; thus this fluorescent metabolite accumulates to high levels in wells with dead cells by 2 to 4 days. A similar increase in fluorescence associated with cytotoxicity was observed in primary cultures of rat hepatocytes using the long-term resazurin-based assay. In addition to an improved signal relative to the short-term assay, the inversion of the fluorescent signal from high = alive short-term to high = dead long-term allows determination of two independent cytotoxicity endpoints after addition of one innocuous vital dye. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.     

Studies on mouse liver urate oxidase

Summary The localization of urate oxidase in mouse liver was investigated by fluorescent antibody technique. The fluorescence was observed in the cytoplasm and fine granules scattering throughout the cytoplasm of liver cells. The diffuse cytoplasmic fluorescence around the central vein was somewhat stronger than that of the medial and outer zone of hepatic lobule. Nuclei of the liver cell and stellate cell of Kuppfer were not stained.     

The histochemical distribution of protein bound sulfhydryl groups in human epidermis by the new staining method.

Recently, we synthesized a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) which is nonfluorescent by itself but will react readily with -SH groups to form highly fluorescent addition products. By the use of this reagent, we studied the localization and concentration of -SH groups and S--S linkages in the human epidermis. The distribution of -SH groups in living layers was abundant in cytoplasm but not in nuclei. The fluorescence was concentrated on the cell membrane or intercellular spaces (MIC parts) and was increased at the spino-granular junction. In the horny layer, the fluorescence of the MIC parts appeared brilliantly in the lower layers and decreased gradually. On the other hand, the fluorescence of cytoplasm in keratinized cells in the stratum corneum was faint. The localization of S--S linkages was not a characteristic of the living layers, but appeared abruptly at the junction of living and horny layers. The fluorescence was localized to the MIC parts and disappeared gradually. The distribution of S--S linkages appeared to be very low in the cytoplasm of keratinized cells. No substantial fluorescence was localized on keratohyalin granules even after reduction.     

Microfluorometric estimates of proteins associated with murine hepatocyte and thymocyte nuclei, residual structures, and nuclear matrix derivatives

Isolated diploid hepatocyte and thymocyte nuclei and their derivatives ("residual structures" and nuclear matrices, as defined by Kaufmann et al. 1981) were evaluated by microfluorometry following reaction with the following fluorochromes: brilliant sulfaflavine (BSF) used at pH 2.8 for the demonstration of total protein; acridine orange (AO) used at pH 9.0 to reveal acidic groups of proteins; and 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin (CPM) used under conditions required to demonstrate the sum of sulfhydryl (SH) and disulfide (SS) groups of proteins. The results suggested that the proteins reacting with AO and CPM differed from each other and from those revealed by fluorochroming with BSF. In every comparison, hepatocyte nuclei and their derivatives were more fluorescent than the respective populations of thymocyte nuclei and their derivatives. In material fluorochromed with BSF and AO, nuclear matrices were less fluorescent than residual structures, which, in turn, were less fluorescent than intact nuclei. In contrast, nuclear matrices fluorochromed with CPM were less fluorescent than intact nuclei but more fluorescent (paradoxically) than residual structures. The ratios of the total fluorescence values of hepatocyte and thymocyte nuclei fluorochromed with BSF changed significantly during extractions required to produce residual structures and nuclear matrices, while comparable ratios in material fluorochromed with AO or CPM did not change significantly. Comparisons of the ratios of the fluorescence values of intact nuclei and their derivatives in a variety of combinations yielded complex and variable results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aminergic Innervation of the Cranial and Caudal Neurosecretory Systems in the Teleost Gillichthys mirabilis

Abstract Numerous fluorescent varicosities surround most of the caudal neurosecretory neurons and also regularly occur among pars intermedia cells of the adenohypophysis in the teleost, Gillichthys mirabilis. The color of the varicosities, as well as their responses to pharmacological treatments, is diagnostic of catecholaminergic neurons and processes. No fluorescence characteristic of monamines is found in the rostral pars distalis, in the proximal pars distalis or in the cells of the nucleus lateralis tuberis (NLT), although fluorescent varicosities are found within the ventral hypothalamus in the vicinity of the NLT. Bilateral clusters of fluorescent cell bodies are located in the ventral hypothalamus (posterior to the NLT); some of these cells border the neurohypophysis. Fluorescent tracts from these cell clusters extend to a pair of elongate nuclei of nonfluorescent neurons which are surrounded by fluorescent varicosities. Alteration of osmotic conditions did not effect the fluorescence, except for the caudal neurosecretory cells of fish exposed to fresh water for long periods. Adrenergic nervous input thus seems to be an important component of both the cranial and caudal neurosecretory systems.     

Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein

The unique morphology and cell-specific expression of surfactant genes have been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells on the basis of transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter. Green fluorescent cells that colocalized by immunostaining with endogenous pro-SP-C were scattered throughout the parenchyma. EGFP was not detected in Clara cell secretory protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-C immunostaining diminished in lungs exposed to hyperoxia, consistent with decreased expression and secretion of intracellular precursor protein. In contrast, type II cells could still be identified by their intrinsic green fluorescence, because EGFP is not secreted. Type II cells could also be purified from single-cell suspensions of lung homogenates using fluorescence-activated cell sorting. Less than 1% of presorted cells exhibited green fluorescence compared with >95% of the sorted population. As expected for type II cells, ultrastructural analysis revealed that the sorted cells contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected in the sorted population, but T1alpha and CD31 (platelet endothelial cell adhesion molecule) were not, indicating enrichment of type II epithelial cells. This method will be invaluable for detecting and isolating mouse type II cells under a variety of experimental conditions.     

Fluorogen-activating single-chain antibodies for imaging cell surface proteins

Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.     

Intracellular translocation and metabolism of a fluorescent phosphatidic acid analogue in cultured fibroblasts

We have investigated the metabolism and intracellular translocation of a fluorescent derivative of phosphatidic acid, 1-acyl-2-[(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl] phosphatidic acid (C6-NBD-PA), and its metabolites, in Chinese hamster fibroblasts. This derivative is rapidly transferred from phospholipid vesicles to cells at 2 degrees C, and results in fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear membrane of intact cells during its metabolism predominantly to fluorescent diglyceride (Pagano, R. E., Longmuir, K. J., Martin, O. C., and Struck, D. K. (1981) J. Cell Biol. 91, 872-877). In the present study, we show that, upon warming to 37 degrees C, the fluorescence associated with the endoplasmic reticulum was greatly reduced, while cytoplasmic lipid droplets, which were initially nonfluorescent, became intensely labeled. This altered intracellular distribution of fluorescence was accompanied by further metabolism of the fluorescent lipids to NBD-triglyceride and NBD-phosphatidylcholine. Although NBD-fatty acid was also produced, it was not re-utilized in the synthesis of other cellular lipids. Subcellular fractionation experiments demonstrated that primarily NBD-labeled triglyceride was associated with the intracellular lipid droplets, although substantial amounts of NBD-labeled phosphatidic acid, phosphatidylcholine, and diglyceride were also present in the whole cell extracts. This finding was confirmed in a separate experiment in which the fluorescent lipids associated with the intracellular lipid droplets were selectively and irreversibly photobleached in situ. Extraction and analysis of the fluorescent lipids revealed that NBD-triglyceride was preferentially photobleached. These results indicate that "sorting" of the NBD-labeled lipids into various cytoplasmic compartments accompanied their metabolism.     

An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio

Protein-protein interactions (PPIs) play crucial roles in various biological processes. Among biochemical, genetic, and imaging approaches that have been used for the study of PPIs, visualization of PPIs in living cells is the key to understanding their cellular functions. The bimolecular fluorescence complementation (BiFC) assay represents one of these imaging tools for direct visualization of PPIs in living cells. The BiFC assay is based on the structural complementation of two nonfluorescent N- and C-terminal fragments of a fluorescent protein when they are fused to a pair of interacting proteins. Although over 10 different fluorescent proteins have been used for BiFC assays, the two nonfluorescent fragments from all of these fluorescent proteins can spontaneously self-assemble, which contributes to background fluorescence and decreases the signal-to-noise (S/N) ratio in the BiFC assay. Here we report the identification of a mutation, I152L, that can specifically reduce self-assembly and decrease background fluorescence in a Venus-based BiFC system. This mutation allows a 4-fold increase in the S/N ratio of the BiFC assay in living cells. This improved Venus-based BiFC system will facilitate PPI studies in various biological research fields.