Use Of Hoechst Research Articles

Sex preselection: Laboratory validation of the sperm sex ratio of flow sorted X- and Y-sperm by sort reanalysis for DNA

Laboratory validation is essential in developing an effective method for separating X and Y sperm to preselect sex. Utilizing sexed sperm from a particular experiment to test fertility and achieve the subsequent phenotypic sex without knowing the likely outcome at conception is too costly for most applications. Further, research advances need to be built on an ongoing assessment with respect to the collection of data to continue progress towards achieving a successful outcome. The Beltsville Sperm Sexing Technology, which is based on the sorting of X- and Y-bearing sperm through the process of flow-cytometric sperm sorting, is also well suited for validation in the laboratory by “sort reanalysis” of the sperm X- and Y-bearing fractions for DNA content. Since the sexing technology is based on the use of Hoechst 33342, a permeant nuclear DNA stain for sorting X- and Y-bearing sperm, it also can be the marker for determining the proportions of X and Y populations by sort reanalysis. The process consists of using an aliquot of the sorted sperm and sonicating to obtain sperm nuclei. The uniformity of the nuclear staining is re-established through the addition of more Hoechst 33342. Separate analysis of each aliquot produces a histogram that is fitted to a double gaussian curve to determine proportions of X and Y populations. The relative breadths of the distributions of DNA of X- and Y-bearing sperm within a species affects interpretations of the histogram. Sort reanalysis is consistently repeatable with differences in X/Y DNA equal to or greater than 3.0%. This information on sex ratio of the sperm then provides the precise tool by which one can predict the outcome in terms of sex, from a particular sample of semen. Simple analysis of unsorted sperm to determine the proportions of X- and Y-bearing sperm based on DNA content is also an effective tool for validating sperm-sex ratio, whether it is in a sample assumed to be 50:50 or predicted to be something other than 50:50. This simple analysis provides for a check on the potential sex ratio of any sample of semen.

Use of Hoechst 33342 stain to evaluate live fresh and frozen bull sperm by computer-assisted analysis.

The objective of this research was to investigate possible procedures for evaluating living bull sperm stained with Hoechst 33342 while in a simple medium and in commonly used complex egg yolk-glycerol-Tris (EYGT) and whole milk-glycerol (WMG) extenders. The two semen extenders provide good cryoprotection, but the latter one virtually obscures the sperm. To evaluate sperm motion characteristics when static nonsperm particles are present, a new Hamilton Thorne epifluorescent optical system (UV) with a strobe light was developed for potential use with DNA-stained sperm. This system permitted examination for the first time of sperm motion characteristics in milk. In Experiment 1 (four bull semen replicates with five dye concentrations and three incubation times), 2.5 microg/ml of Hoechst 33342 stained live and dead sperm sufficiently in a modified Tyrode's solution to measure all sperm characteristics without depressing motility, which was validated by using phase-contrast to analyze stained and unstained controls. In Experiments 2a and 2b, each using semen from four bulls with a 5 x 5 factorial arrangement, it was determined that 40 to 60 microg/ml of dye in EYGT or WMG, with UV illumination for 20 minutes, was optimal. There was no detrimental effect on sperm motility. In Experiment 3, analyses of two ejaculates, from each of eight bulls, confirmed that motion characteristics of sperm in EYGT and WMG were not depressed when the sperm were stained with Hoechst 33342. These experiments demonstrate that the dye concentrations and exposure times developed for use with the new epifluorescent optics facilitate evaluating bull sperm frozen in particle-filled whole milk and should be useful for sperm evaluation of a variety of species when nonsperm particulate matter may otherwise interfere.

Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord.

The infusion of BDNF and NT-3 into Schwann cell (SC) grafts promotes regeneration of brainstem neurones into the grafts placed in adult rat spinal cord transected at T8 (Xu et al., 1995b). Here, we compared normal SCs with SCs genetically modified to secrete human BDNF, grafted as trails 5 mm long in the cord distal to a transection site and also deposited in the transection site, for their ability to stimulate supraspinal axonal regeneration beyond the injury. SCs were infected with the replication-deficient retroviral vector pL(hBDNF)RNL encoding the human preproBDNF cDNA. The amounts of BDNF secreted (as detected by ELISA) were 23 and 5 ng/24 h per 106 cells for infected and normal SCs, respectively. Biological activity of the secreted BDNF was confirmed by retinal ganglion cell bioassay. The adult rat spinal cord was transected at T8. The use of Hoechst prelabelled SCs demonstrated that trails were maintained for a month. In controls, no SCs were grafted. One month after grafting, axons were present in SC trails. More 5-HT-positive and some DbetaH-positive fibres were observed in the infected vs. normal SC trails. When Fast Blue was injected 5 mm below the transection site (at the end of the trail), as many as 135 retrogradely labelled neurones could be found in the brainstem, mostly in the reticular and raphe nuclei (normal SCs, up to 22, mostly in vestibular nuclei). Numerous neurones were labelled in the ventral hypothalamus (normal SCs, 0). Also, following Fast Blue injection, a mean of 138 labelled cells was present in dorsal root ganglia (normal SCs, 46) and spinal cord (39 vs. 32) rostral to the transection. No labelled spinal neurones rostral to the transection were seen when SCs were not transplanted. Thus, the transplantation of SCs secreting increased amounts of BDNF improved the regenerative response across a transection site in the thoracic cord. Moreover, the enhanced regeneration observed with infected SCs may be specific as the largest response was from neurones known to express trkB.

Sequence-Specific Gene Detection with a Gold Electrode Modified with DNA Probes and an Electrochemically Active Dye

A synthesized 20-mer DNA probe complementary to a part of an oncogene v-myc region having a mercaptohexyl group at the 5'-phosphate end was immobilized on a gold electrode by chemisorption. The immobilized DNA was detected voltammetrically using Hoechst 33258 with a DNA minor groove binder and an electrochemically active dye. The modified electrode was immersed into a 100 mumol/L Hoechst 33258 solution and washed with a phosphate buffer (pH 7.0). The anodic peak current (ipa) of Hoechst 33258 on the modified electrode was higher than that on a bare gold electrode (128 and 75 nA, respectively). It was considered that Hoechst 33258 was concentrated on the electrode surface due to its association with DNA. When the modified electrode was hybridized in a solution of a model targeted gene (10(-7) g/mL), single-stranded pVM623 containing the PstI fragment of a 1.5-kilobase pair of oncogene v-myc, the ipa was 192 nA. On the other hand, the ipa was 128 nA when the modified electrode was reacted in a solution of single-stranded pUC119 without a region complementary to v-myc in pVM623. The ipa was related to the concentration of the targeted DNA in the hybridization reaction. The use of Hoechst 33258 resulted in a sequence-specific detection of the targeted DNA quantitatively ranging from 10(-7) to 10(-13) g/mL in a buffer solution.

TUMOR VASCULAR SHUTDOWN FOLLOWING PHOTODYNAMIC THERAPY BASED ON POLYHEMATOPORPHYRIN OR 5-AMINOLEVULINIC ACID

The effects of photodynamic therapy (PDT) based on polyhaematoporphyrin (PHP) or 5-aminolaevulinic acid (ALA) on the tumour vascular perfusion (TVP) of a rat fibrosarcoma were examined using an (RbCl)-Rb-86 extraction technique. Both forms of therapy induced large and highly significant reductions in TVP, countering the previous suggestion that ALA-PDT has no vascular effects. Ultrasensitive digital imaging fluorescence microscopy indicated that ALA-induced PpIX was distributed relatively evenly throughout the tumour. Use of Hoechst 33342 as a vascular marker revealed that PpIX was also present in cells lining the tumour microvascular spaces, contrary to previous findings.

The use of Hoechst 33342 to stain donor nuclei used in the bovine nuclear transfer procedure

The use of Hoechst 33342 to stain donor nuclei used in the bovine nuclear transfer procedure

Dynamics of chromatin changes in live one-cell mouse embryos: A continuous follow-up by fluorescence microscopy

Conditions of minimal dye concentration and minimal irradiation which allow the continuous observation of pronuclei in live unicellular mouse eggs by fluorescence microscopy have been found with the use of Hoechst 33342 as fluorophore and a camera of high sensitivity coupled with an image processing system allowing true integration of weak fluorescent signals and further treatment and analysis. Under these conditions the developmental potential of the embryos is not affected. Using such an approach, which avoids eventual artifacts due to fixation procedure, we describe the changes in the nuclear organization and chromatin structure, from formation of pronuclei to mitosis, with particular attention to the chromatin associated with nucleoli and the timing process of chromatin condensation.

Application of pyronin Y(G) in cytochemistry of nucleic acids

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y(PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation. The products of PY interaction with double-stranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry. PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 microM, the RNA stainability decreases, perhaps due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in G2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.

HOECHST STAINING AND QUANTIFICATION OF SPERM IN THE SPERMATOPHORE AND SPERMATHECA OF THE DECORATED CRICKET,GRYLLODES SUPPLICANS(ORTHOPTERA: GRYLLIDAE)

AbstractThe use of Hoechst (33258) stain in quantifying spermatozoa contained in the spermatheca and spermatophore of the decorated cricket,Gryllodes supplicans(Walker) (Orthoptera: Gryllidae), is described. The technique was used to estimate the number of sperm remaining in spermatophores removed from females at various times after females had mated.

Fluorometric Determination of DNA in Aquatic Microorganisms by Use of Hoechst 33258

A method for the determination of microbial DNA in aquatic environments by the use of Hoechst 33258 has been developed. With unsophisticated instrumentation and simple extraction procedures, it is possible to detect from 0.05 to 10 mug of DNA in bacterial cultures or natural water samples. The method is specific for DNA; DNase I treatment of extracts of natural microbial populations removed 95 to 100% of the observed fluorescence. DNA content ranged from 165 ng ml for relatively eutrophic Potomac River water to 27 ng ml for coastal Atlantic Ocean water and was correlated to an acridine orange direct count (r = 0.90).

Use of Hoechst 33342 for cell selection from multicell systems.

Hoechst 33342 staining of multicell spheroids, three-dimensional cell clusters grown in vitro, results in a marked gradient of cellular fluorescent intensities inward from the spheroid periphery. The penetration of the dye is concentration and time dependent, so staining can be coupled with fluorescence activated cell sorting techniques to allow disaggregated single cells to be sorted or selected according to their degree of staining and therefore their depth within the spheroid. We have found the staining procedure to be highly reproducible, and to result in minimal toxicity even to the more brightly staining external cells. Comparison of this technique with others for cell selection suggests that increased resolution is available with the Hoechst technique.

Fluorometric determination of DNA in aquatic microorganisms by use of Hoechst 33258 : Paul, J.H. and Brenda Myers, 1982. Appl. environ. Microbiol., 43(6): 1393–1399

Fluorometric determination of DNA in aquatic microorganisms by use of Hoechst 33258 : Paul, J.H. and Brenda Myers, 1982. Appl. environ. Microbiol., 43(6): 1393–1399