Lysis Solution Research Articles

Long-Term Preservation of Whole Blood Samples for Flow Cytometry Analysis in Normal and HIV-Infected Individuals from Africa

A whole blood method requiring less than 4 ml of heparinized blood was developed to assess the practicality of preparing whole blood samples that could be easily stored, transported and readily used to determine the lymphocyte phenotypes and proliferation responses of individuals from remote areas who are infected with the human immunodeficiency virus. Minor modifications in standard whole blood procedure for lymphocyte phenotyping have significantly increased the stability of light scatter and fluorescence intensity of the cells for subsequent flow cytometry (FC) analysis. These changes include removal of lysis solution prior to fixation, fixation of monoclonal antibody-stained cells in 1% paraformaldehyde for 30 minutes and storage of fixed samples in medium containing 1% bovine serum albumin. Lymphocyte subsets and their functional subsets could reliably be determined on samples stored for up to 4 weeks. Further, blood samples could be kept at room temperature for up to 96 hours or at ambient temperature during transportation from Africa before staining for FC without affecting their quantitation. While samples could be processed for FC analysis under field-laboratory conditions, proliferation assays could only be performed on samples that were transported within 48 hours of their collection. The whole blood method saves time and expense and decreases the volumes of blood required to perform phenotypic analysis and functional assays on specimens collected in remote areas.

Rapid nucleic acid assay for detection of bacteria with tetM-mediated tetracycline resistance

A novel nucleic acid assay has been developed to screen bacterial populations for the presence of the tetM structural gene. The method involves the specific hybridization of several synthetic oligonucleotides to the gene in a crude bacterial lysate solution. As few as 1.5 x 10(4) CFU can be detected with the assay.

Effect of nickel(II) on DNA-protein interactions

Alterations in DNA-protein interactions (DPI) may play an important role in carcinogenesis. Although the mechanism of nickel carcinogenesis is unknown, nickel reportedly affects DPI. A microfiltration, nitrocellulose filter assay was utilized to study DPI in intact Chinese hamster ovary (CHO) cells and in isolated nuclei. Prior to exposure of CHO cells or isolated CHO cell nuclei, DNA and proteins were radiolabeled using 3H-thymidine and 35S-methionine, respectively. Nuclei were exposed to NiCl2 in 10 mM HEPES buffer (pH 6.8). CHO cells were exposed in either complete or a salts-glucose medium. Following exposure, nuclei or cells were incubated at 37 degrees C for 20 min in a high salt lysis solution; aliquots were loaded onto nitrocellulose filters and washed with a low salt solution. DNA (3H) retained on each filter was normalized to protein (35S) bound on the filter. Exposure of either whole cells or isolated nuclei to increasing, noncytotoxic concentrations of NiCl2 resulted in a dose dependent decrease in DPI. The effect of nickel on specific DNA-protein interactions was examined using a band shift assay and a cloned satellite DNA sequence. Nickel inhibited specific protein binding to the satellite DNA probe. The results of these two independent assays, which were conducted at physiological pH, indicate that NiCl2 inhibits specific DNA-protein interactions.

Linear DNA Elution Dose Response Curves Obtained in CHO Cells with Non-unwinding Filter Elution after Appropriate Selection of the Lysis Conditions

The effect of detergent type, pH and temperature during lysis on the DNA elution dose response was studied under non-winding conditions in exponentially growing, plateau-phase and synchronous S-phase CHO cells. Lysis with sodium-N-laurylsarcosine (NLS) increased the DNA elution rate and resulted in higher DNA elution for the same absorbed radiation dose than lysis with sodium dodecyl sulphate (SDS). This increase in elution caused a reduction in the shoulder width of the DNA elution dose-response curve, but did not significantly affect the final slope. One hour incubation at elevated temperatures (60 degrees C) during lysis either with NLS or SDS further increased DNA elution. Under these conditions DNA elution dose-response curves with a small or zero shoulder were obtained with exponentially growing, plateau-phase or synchronized S-phase cells. DNA elution was reduced to about 50 per cent of the controls when the pH of the SDS lysis solution was adjusted from 9.6 to 7.6. This effect was observed in cells that were lysed at room temperature, as well as in cells lysed at 60 degrees C. When NLS was used for lysis, a similar reduction in pH did not alter the DNA elution dose-response curve at either lysis temperature. Based on these results it is suggested that the shoulder observed in the DNA elution dose-response curve reflects partial separation of DNA from associated proteins. A direct and unconditional correlation of the DNA filter elution behaviour, as observed under non-unwinding conditions, with the induction of DNA dsb may thus not always be justified. Caution is required when elution results are used to establish correlations between the level of induction of DNA dsb and cell killing.

EDTA alkaline elution characteristics and measurement of DNA damage in unlabeled DNA using hoechst 33258 fluorescence

Hoechst 33258 fluorescence of single stranded DNA has been used to perform alkaline elution with unlabeled DNA. The high background fluorescence of "standard" elution solutions has prompted others to use EDTA but the elution characteristics of DNA in EDTA-containing solutions and the comparability of results with those using "standard" tetrapropyl ammonium hydroxide solutions have not previously been examined. We report here the elution characteristics of DNA in EDTA and the relevant parameters for the successful use of EDTA as an elution solution. An increase in elution pH to 12.4 is required but elution solutions of higher pH cause alkaline hydrolysis of undamaged DNA. Drug-treated DNA from which DNA-protein crosslinks have been removed can be completely removed from the filters at the end of the elution by a Pronase filter digestion. The simplest and most efficient removal of DNA-protein crosslinks is through the inclusion of proteinase-K in an SDS containing lysis solution. EDTA elution can measure interstrand crosslinks and single strand breaks as easily as is performed using radiolabeled DNA under "standard" elution conditions and requires only 1.5-2 x 10(6) cells per elution filter. DNA-protein crosslinking measurements were unsatisfactory, however, since even the Pronase digestion failed to completely remove protein-crosslinked DNA from the elution filters.

The Induction of DNA-Protein Crosslinks in Hypoxic Cells and Their Possible Contribution to Cell Lethality

The induction of single-strand breaks (SSBs) in the DNA of Chinese hamster ovary cells by X rays under different irradiation conditions was measured by the alkaline elution technique. The oxygen enhancement ratio (OER) for SSB induction determined for cells irradiated in air versus irradiation of cells made hypoxic by metabolic depletion of O2 was 9.7. However, when proteinase K was included in the cell lysis solution the OER was reduced to 4.2. The proteinase affected the elution rate only of the cells irradiated under hypoxic conditions, suggesting that DNA-protein crosslinks (DPCs) are preferentially produced in hypoxic cells by radiation. The ability to repair these DPCs was compared in two cell lines: the wild-type AA8 line and an excision-repair-deficient mutant line, UV-41. The AA8 line removed about 80% of the DPCs induced by radiation under hypoxic conditions within a 24-h repair incubation. The UV-41 line, on the other hand, removed only about 20% of the DPCs in the same time. The OERs for cell survival of these two lines are 3.1 for AA8 but only 1.9 for UV-41, suggesting that the DPCs preferentially induced in the DNA of cells irradiated under hypoxic conditions may contribute to cell killing when the normal DNA-repair mechanisms are compromised.

The quantitation of actin in human lymphocytes by isoelectric focusing

A simple and sensitive technique for quantitating the actin content of lymphocytes is described. The method uses isoelectric focusing over a pH gradient of 4.5 to 6.5 to resolve actin from other polypeptides followed by densitometric scanning of Coomassie-blue stained gels. The dye uptake of purified rabbit-skeletal-muscle actin standards was linear from 0.5 to 5.0 μg of applied protein. Cell protein was solubilized in a lysis solution containing urea, β-mercaptoethanol, and NP40. Linear results were obtained when homogenates prepared from 5 to 25 × 10 5 cells were applied. The actin content of human peripheral blood lymphocytes determined with this assay was 2.1 ± 0.3 mg/10 9 cells and represented 6.3 ± 1.6% of the total cellular protein. The ratio of β:γ actin in human lymphocytes was 2:1.

Alkali-sensitive sites in DNA from human cells treated with ultraviolet light, 1'-acetoxysafrole or 1'-acetoxyestragole.

The formation and repair of alkali-labile sites in the DNA of human cells treated with 254 nm u.v. light, 1'-acetoxyestragole (1'-AcO-E) or 1'-acetoxysafrole (1'-AcO-S) have been studied. DNA was analysed by sedimentation in alkaline sucrose gradients after the cells had been layered on the gradients in lysis solution for 15 h (long lysis) or for only 0.75 h (short lysis). With the long lysis technique, a dose of 20 J/m2 resulted in 0.2-0.4 strand breaks/10(8) daltons while treatment of cells with 0.5 mM 1'-AcO-E or 1'-AcO-S caused 0.1-0.3 strand breaks/10(8) daltons. In excision repair proficient T98G cells, one third to two thirds of these strand breaks disappeared upon 4 h incubation after exposure to each of the three agents. In excision repair deficient xeroderma pigmentosum fibroblasts (XPA), the alkali-labile sites produced by 1'-AcO-E or 1'-AcO-S were still repaired, although those resulting from u.v.-irradiation were not. Similar characteristics were observed after the short lysis period. The sedimentation velocities of nucleoids, prepared from treated XPA cells, in neutral sucrose gradients containing ethidium bromide, did not reveal the presence of overt strand breaks in the DNA, suggesting that the lesions were of a type in which the sugar-phosphate backbone was intact but sensitive to hydrolysis by alkali. The contribution of this type of damage to the total DNA damage produced by the agents was estimated to be less than 1% for u.v., and less than 2.5% for 1'-AcO-E and 1'-AcO-S.

Fluorometric determination of DNA and RNA in Chlamydomonas using ethidium bromide.

By using the fluorescence enhancement of ethidium bromide bound to nuclei acid, a very rapid, simple and sensitive assay of DNA in the green alga Chlamydomonas has been devised. Total fluorescence (DNA + RNA) was determined by complex formation with ethidium bromide in a cell lysate made by mixing cell samples with lauroyl sarcosinate, EDTA and NaOH and incubating the mixture for 5 min at room temperature followed by neutralization. For determination of DNA the RNA was digested by incubating the cell sample in te alkaline lysis solution for 45 min at 60 degrees C followed by neutralization, and complex formation with ethidium bromide. Quenching of the fluorescence due to cellular pigments was corrected for using an internal DNA standard.

The nitrite methemoglobin complex—Its significance in methemoglobin analyses and its possible role in methemoglobinemia

Evidence consistent with the formation of a reversible complex between excess free nitrite ion and the ferric heme groups of methemoglobin is presented. The existence of this complex in nitrited red cells or in lysates in the presence of appropriate concentrations of free nitrite can lead to gross underestimation of methemoglobin levels by a common spectrophotometric method. Complex formation is manifested by a decrease in the absorption of methemoglobin solutions in the range of 600–650 mμ. This source of error can be eliminated in red cell suspensions simply by washing. Similarly, lysate solutions may be dialyzed or diluted to effect dissociation of the complex. Attempts were made to estimate the dissociation constant of this complex at 25°, pH 7.4, and physiologic ionic strength. Although unknown side reactions between methemoglobin and nitrite interfere in the spectrophotometric determination of this constant, its value is probably not greater than 3 mM. This estimate indicates that the complex must exist transiently in vivo when mice receive nitrite doses adequate to convert more than one-third of the circulating blood pigment to methemoglobin. Thus, complex formation may contribute to the unusually sustained methemoglobinemia produced in mice by nitrite.