A quantitative microassay of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on polystyrene plates.

A microadhesion assay that allows the quantitative determination of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on 96-well polystyrene plates has been developed. After dislodging nonadherent cells by centrifugation, specifically bound cells are quantified by colorimetric analysis of a blue formazan product generated from the dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide by enzymatic reduction. Carbohydrate specificity of the cell adhesion was demonstrated by inhibition analyses and the general applicability of the assay was proved with indicator cells of three different origins: mouse fibrosarcoma cells, Chang liver cells, and human breast carcinoma cells (MDA-MB 231).     

Sulforhodamine B colorimetric assay for cytotoxicity screening

The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening.     

A low-cost flow cytometric assay for the detection and quantification of apoptosis using an anionic halogenated fluorescein dye

We describe here a technical improvement of an established colorimetric method used to detect and measure the occurrence of apoptosis in mammalian cells during in vitro cell culture. This assay uses an anionic halogenated fluorescein dye that is taken up by apoptotic cells at the stage of phosphatidylserine externalization. We demonstrate that apoptotic cells stained with this dye can be detected by flow cytometric analysis. Furthermore, we show that the modified method compares well with the standard annexin-V-based apoptosis assay and that it is significantly more cost-effective than the annexin-V assay.     

Quantification of neutral red pinocytosis by small numbers of adherent cells: comparative studies.

A colorimetric assay has been developed for studies on neutral red (NR) pinocytosis by small numbers (below 2 x 10(5)) of adherent cells cultured in 96 well plates. The NR uptake per cell mass was much higher in the sea urchin perivisceral adherent cells and human HL-60 cell line monolayers than in the murine and Atlantic salmon macrophages. The apparent difference points to the usefulness of this novel assay in comparative studies.     

A dye binding method for measurement of total protein in microalgae

Protein is a large component of the standing biomass of algae. The total protein content of algae is difficult to measure because of the problems encountered in extracting all of the protein from the cells. Here we modified an existing protein assay to measure total protein in microalgae cells that involves little or no extraction of protein from the cells. Aliquots of fresh or pretreated cells were spotted onto filter paper strips. After drying, the strips were stained in a 0.1% (w/v) solution of the protein stain Coomassie Brilliant Blue R-250 for 16 to 24 h and then destained. The stained protein spots were cut out from the paper, and dye was eluted in 1% (w/v) sodium dodecyl sulfate (SDS). Absorbance at 600 nm was directly proportional to protein concentration. Cells that were recalcitrant to taking up the dye could be either heated at 80°C for 10 min in 1% SDS or briefly sonicated for 3 min to facilitate penetration of the dye into the cells. Total protein measured in Chlorella vulgaris using this method compared closely with that measured using the total N method. Total protein concentrations were measured successfully in 12 algal species using this dye binding method.     

Neutral red uptake assay for the estimation of cell viability/cytotoxicity

The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.     

Spectrophotometric quantitation of anchorage-dependent cell numbers using extraction of naphthol blue-black-stained cellular protein

A convenient method is described by which the actual or relative number of cells in anchorage culture is determined. After removal of the growth medium, cells are subjected to a double-fixation procedure. The cellular protein content is subsequently quantitatively stained with naphthol blue-black. After a period of removal of unbound stain, dye-protein complexes are hydrolytically released and measured spectrophotometrically at 620 nm. A linear correlation exists (r = 0.994) between cell concentration, in the range 3 X 10(4) to 8 X 10(5) cells/ml of final assay volume, and absorbance up to reading values of 3.8. The technical reproducibility of the assay, as judged from assessments of cell numbers in suspension culture, displays a coefficient of variation of 5%. The method was developed for 9.6-cm2 culture dishes, but it should be possible to transform it for the use of microtiter plates.     

Colorimetric Assay System for Screening Antiviral Compounds against Hepatitis B Virus

A highly sensitive, rapid, and accurate assay system was developed for the in vitro evaluation of anti-hepatitis B virus (anti-HBV) agents. Chronic HBV-producing HB611 cells were used in combination with immunoaffinity purification, polymerase chain reaction (PCR), and hybrid capture detection. HB611 cells were incubated with putative anti-HBV agents for 7 days in 96-well microtiter plates. HBV was purified from HB611 cell culture media using immunoaffinity purification. The HBV DNA was extracted, amplified with PCR, and assayed using a hybrid capture colorimetric method. This assay provided quantitative detection of extracellular HBV DNA from 25 μl of cell culture media. Using the colorimetric method, we found that 50% effective concentration levels of several known anti-HBV agents (HPMPA, PMEDAP, PMEA and others) were similar to those reported in studies using Southern blot analysis. These results demonstrate that this new and easily automated colorimetric assay system can be used for the rapid and accurate assessment of anti-HBV compound selectivity.     

Basic Two-Dye Stains for Epoxy-Embedded 0.3-1 μ Sections

Dyes used in the 3 methods recommended are: I, thionin and acridine orange (T-AO); II, Janus green and Darrow red (JG-DR); III, methyl green and methyl violet (MG-MV). The first 2 methods were two-solution stains, applied in sequence; the third, required only one solution since methyl violet is present in commercial methyl green. Staining solution and timing was as follows: Method I. 0.1% thionin in a 45% ethanolic solution of 0.01 N NaOH, 5 min at 70 C; rinsing in water and followed by 1 min in a 1% aqueous solution of acridine orange made up in 0.02 N NaOH, also at 70 C, then washed, and dried on slides. Method II. 0.5% Janus green in aqueous 0.05 N NaOH, 5 min at 70 C; rinsing in water then into 0.5% Darrow red in 0.05 N NaOH (aq.), 2 min at 70 C., washing, and drying on slides. Method III. 1% methyl green (commercial, unpurified) in 1% aqueous borax for 15-20 min at 20-25 C, washing and attaching to slides. All staining was performed by floating the sections on the staining solutions, all drying at 70 C, and mounting in a resinous medium. T-AO gave blue to violet cytoplasmic structures, darker nuclei which contrasted strongly with yellow connective tissue and the secretion of goblet cells. JG-DR resembled a hematoxylineosin stain, but by shortening the staining time in DR to 0.5-1 min, collagenous and elastic tissue retained more of the green dye. MG-MV gave dark green nuclei in light green cytoplasm, with collagenous and elastic tissues in blue to violet. As with most methods for staining ultrathin sections, thicknesses of less than 1 μ required longer staining times.     

Differential Dichrome Staining of Tissue Culture Monolayers: Alternate Dyes and Possible Mechanism

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Differential dichrome staining of tissue culture monolayers: alternate dyes and possible mechanism.

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Parabolic growth patterns in 96-well plate cell growth experiments

Summary In preparing for the routine use of the ubiquitous in vitro cell growth inhibition assay for the study of anticancer agents, we characterized the statistical properties of the assay and found some surprising results. Parabolic well-to-well cell growth patterns were discovered, which could profoundly affect the results of routine growth inhibition studies of anticancer and other agents. Four human ovarian cell lines, A2780/WT, A2780/DX5, A2780/DX5B, and A121, and one human ileocecal adenocarcinoma cell line, HCT-8, were seeded into plastic 96-well plates with a 12-channel pipette, without drugs, and grown from 1–5 d. The wells were washed with a plate washer, cells stained with sulforhodamine B (SRB), and dye absorbance measured with a plate reader. Variance models were fit to the data from replicates to determine the nature of the heteroscedastic error structure. Exponential growth models were fit to data to estimate doubling times for each cell line. Polynomial models were fit to data from 10-plate stacks of 96-well plates to explore nonuniformity of cell growth in wells in different regions of the stacks. Each separate step in the assay was examined for precision, patterns, and underlying causes of variation. Differential evaporation of water from wells is likely a major, but not exclusive, contributor to the systematic well-to-well cell growth patterns. Because the fundamental underlying causes of the parabolic growth patterns were not conclusively found, a randomization step for the growth assay was developed.     

A new colorimetric assay for glutathione transferase-catalyzed halogen ion release for high-throughput screening

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes catalyzing the conjugation of a broad range of toxicologically important halogenated compounds to the tripeptide glutathione (GSH) with concomitant halogen ion release. In the present work, a rapid quantitative screening method for GSTs based on colorimetric measurement of halogen ions released from halogenated xenobiotics was developed. The assay is based on the color formation resulting from the reaction of Hg(SCN)₂ with the released halogen ion of the substrate in the presence of Fe³⁺. The color intensity is proportional to the extent of the catalytic reaction, allowing a quantitative measurement of the GST catalytic activity. The assay can be performed using purified recombinant enzyme (the isoenzyme GmGSTU4-4 from Glycine max) or crude recombinant Escherichia coli cell lysates in 96-well microtiter plates. The suitability of the colorimetric assay for screening mutant GST variants derived from a directed evolution library was successfully evaluated. In addition, the assay was also used for screening GST synthetic inhibitors. It was concluded that the proposed colorimetric assay is selective and sensitive and allows the screening of large numbers of samples within a few minutes.     

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.     

FINE STRUCTURE STUDIES OF PHLEUM ROOT MERISTEM CELLS. I. MITOCHONDRIA

Avers , Charlotte J. (Douglass Coll., Rutgers—The State U., New Brunswick, N.J.) Fine structure studies of Phleum root meristem cells. I. Mitochondria. Amer. Jour. Bot. 49(9): 996–1003. Illus. 1962.—Mitochondrial numbers were computed from thick sections embedded in paraffin and stained with iron hematoxylin, from methacrylate embedded, 0.5 μ-thick sections stained with osmium, and from electron micrographs of ultrathin sections fixed in KMnO₄. The mean mitochondrial counts per average cell (20 × 20 × 10μ) were: 121 ± 4 in paraffin sections, 600 ± 40 in methacrylate sections, and 991 ± 96 from electron micrographs. These numbers were higher than the Janus green B count of 91 ± 2 per cell found during an earlier study. In each case, arithmetical conversion factors were used to calculate total numbers of mitochondria per cell since section thicknesses were less than that of whole cell length or depth. Determinations of numbers of mitochondria probably varied depending on section thickness and specific staining procedures used, but the higher count from electron micrographs was assumed to be reasonable on a volumetric basis. The cytoplasmic volume of the average cell is about 2500 μ³ and the volume of a single average mitochondrion is about 0.1μ^3. On this basis, the variety of structures and their relatively sparse distribution seems possible despite the apparently high numbers found. In addition to mitochondria, the numbers of various cellular components per unit cytoplasmic area were determined. These data showed that mitochondria were about 6 times more numerous than proplastids, about 4.5 times more numerous than Golgi bodies, and even more frequent when compared with vesicles, dense bodies, or microbodies. No correlation was found between cytoplasmic area and numbers of organelles per unit area. Photographic evidence was presented for the occurrence in plant cells of the hepatic cell, polymorphic “lysosome” described by Ashford and Porter. The controversial nature of the lysosome is discussed.     

Basic Fuchsin and Picro-Indigo Carmine: A Selective Stain for Cells in Mitosis and for Autoradiography

Selective staining of dividing nuclei is accomplished as follows: paraffin sections, after hydration, are stained 15 min in a saturated aqueous solution of basic fuchsin, washed, then stained 1.5 min in an equal-volumes mixture of indigo carmine saturated in 70% alcohol, and saturated aqueous picric acid. Removal of excess dye with 3 changes of 70% alcohol, dehydration, clearing and covering in a resinous medium completes the process. Nuclei of dividing cells are stained red; cytoplasm and interphase nuclei, light green. This method has been used successfully for determining the mitotic activity of skin, kidney, liver and other rabbit and mouse tissues. Tissue sections previously prepared as autoradiographs may be stained by this method to facilitate the determination of radioactive labeling of mitotic cells.     

The determination of cellular viability of hybridoma cells in microtitre plates: A colorimetric assay based on neutral red

A colorimetric assay utilising Neutral Red (C.I. 50040), a nuclear stain, was developed to determine the cellular viability of hybridoma cells in microtitre plates. A linear correlation (r=0.99) was found to exist between the uptake of Neutral Red by viable cells and the viable cell count determined by Trypan blue exclusion test. The linearity stretched over the range of cell concentrations normal in batch cultures (2–30×10⁴/0.2 ml) with as little as ±6% intra-plate well-to-well variation and ±10.2% inter-assay variation.Microscopical examinations of viable hybridoma cells stained with Neutral Red showed that it was located in the nucleus. The possble bifunctional activity of the Neutral Red assay as a test for cellular viability and estimating the DNA content of hybridoma cells is discussed along with its application in a drug screening programme.     

Differential Staining of Yeast for Purified Cell Walls, Broken Cells, And Whole Cells

Intact yeast cells are Gram positive but broken or disrupted cells are Gram negative. A counterstain with methyl green provides differential staining between cell wall and cytoplasm. The cells and cell fragments are dried on a slide and stained by a standard Gram stain. The preparation is then treated for 5 min with 1% phosphomolybdic acid, washed, and stained 0.5 min with 1% aqueous methyl green (unpurified by CHCl₃ extraction). Under these conditions whole, intact cells are dark purple or black, walls of broken cells and purified walls are light green, and the exposed cytoplasm stains light purple. All fractions can be easily differentiated.     

HISTOCHEMICAL EVIDENCE OF INTRACELLULAR ENZYMATIC HETEROGENEITY OF PLANT MITOCHONDRIA

Avers , Charlotte J., and Earl E. King . (Douglas College, Rutgers U., New Brunswick, N. J.) Histochemical evidence of intracellular enzymatic heterogeneity of plant mitochondria. Amer. Jour. Bot. 47(3) : 220—225. Illus. 1960.–Mitochondrial counts were made of root tip meristem cells of 4 grass species after 4 different treatments, all without fixation, of whole roots. The mean numbers of mitochondria per cell stained with Janus green B were 85—95, and the mean numbers of mitochondria per cell showing cytochrome oxidase activity after the Nadi reaction were 90—95. There was no significant difference between these 2 sets of values for any of the species tested. Counts of cytoplasmic particles active for acid phosphatase showed mean values of 98-108 per cell, which was highly significant in each species when compared with the Janus-green counts. The succinic dehydrogenase system localized with Neotetrazolium showed mean numbers of mitochondria per cell of 42—58, which represented 50—60% of the Janus green-stained mitochondrial populations and were significantly different. The evidence indicated the occurrence of enzymatic variation among mitochondrial complements of individual cells, as well as the existence of at least 2 kinds of cytoplasmic particles. The excess numbers of phosphatase-active particles were considered possibly to be due to the activity of this hydrolase in lysosomes as well as in mitochondria. Thus, the evidence suggested the occurrence of both heterogeneity and multiplicity of cytoplasmic particles in relation to their enzymatic activities.     

Histochemical quantitation of gamma-glutamyl transferase in human leukemia cell lines.

The enzyme gamma-glutamyl transferase (gamma-GT) is involved in many biochemical systems, including the signal transduction of hematopoietic growth factors. Standard colorimetric gamma-GT assays require larger cell numbers than may be obtainable in many cases, such as with highly purified stem-cell populations. To study gamma-GT expression in limited populations, we used a histochemical stain to analyze gamma-GT semiquantitatively in cells of hematopoietic origin. Several human leukemic cell lines, including one with inducible increases in gamma-GT, were stained for gamma-GT and graded 0 through 4+ for the amount of positive granules. The gamma-GT activity demonstrated by this stain was found to be directly proportional to the gamma-GT activity obtained with a colorimetric assay and could be used to calculate approximate gamma-GT activity. This stain therefore provides a useful method for determining gamma-GT activity when limited cell numbers are available.