Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids
2008; Elsevier BV; Volume: 173; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.080414
ISSN1525-2191
AutoresAaron Orozco, Carolina J. Jorgez, Cassandra Horne, Deborah A. Marquez-Do, Matthew R. Chapman, John R. Rodgers, Farideh Z. Bischoff, Dorothy E. Lewis,
Tópico(s)Renal and related cancers
ResumoMicroparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA+ MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies. Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA+ MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies. Microparticles (MPs) circulate in blood and have been associated with pathology in various diseases.1Simak J Gelderman MP Cell Membrane Microparticles in Blood and Blood Products: potentially Pathogenic Agents and Diagnostic Markers.Transfus Med Rev. 2006; 20: 1-26Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar However, the nature and physical characteristics of MPs are understudied. Most studies focus on quantity, origin, and biological activity of MPs, without specific analysis of the inherent organization. During normal placental development, fetal trophoblasts undergo apoptosis, making it plausible that apoptotic MPs could be generated. We propose that MPs generated specifically through apoptosis are a likely source of DNA suitable for PCR-based testing. Three types of membrane-bound vesicles are found in plasma: exosomes, activated MPs, and apoptotic MPs. All three are rich in phospholipids and can be derived from platelets, leukocytes, erythrocytes, or endothelial cells.2Redman CW Sargent IL Microparticles and Immunomodulation in Pregnancy and Pre-Eclampsia.J Reprod Immunol. 2007; 76: 61-67Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar Exosomes, which range in diameter from 40 to 80 nm, are released from activated cells when the membrane of intracellular multivesicular bodies fuses with the plasma membrane. MPs are larger (>100 nm in diameter) and originate from blebbing membranes of either activated cells or cells undergoing apoptosis.3Toth B Lok CA Boing A Diamant M van der Post JA Friese K Nieuwland R Microparticles and Exosomes: impact on Normal and Complicated Pregnancy.Am J Reprod Immunol. 2007; 58: 389-402Crossref PubMed Scopus (63) Google Scholar, 4Thery C Boussac M Veron P Ricciardi-Castagnoli P Raposo G Garin J Amigorena S Proteomic Analysis of Dendritic Cell-Derived Exosomes: a Secreted Subcellular Compartment Distinct from Apoptotic Vesicles.J Immunol. 2001; 166: 7309-7318PubMed Google Scholar, 5Schiller M Bekeredjian-Ding I Heyder P Blank N Ho AD Lorenz HM Autoantigens Are Translocated into Small Apoptotic Bodies During Early Stages of Apoptosis.Cell Death Differ. 2008; 15: 183-191Crossref PubMed Scopus (149) Google Scholar Apoptotic MPs have nuclear proteins as well as nucleic acids.5Schiller M Bekeredjian-Ding I Heyder P Blank N Ho AD Lorenz HM Autoantigens Are Translocated into Small Apoptotic Bodies During Early Stages of Apoptosis.Cell Death Differ. 2008; 15: 183-191Crossref PubMed Scopus (149) Google Scholar Stable and PCR-amplifiable fetal cell-free DNA (cfDNA) has been detected in maternal plasma and used to predict6Leung TN Zhang J Lau TK Chan LY Lo YM Increased Maternal Plasma Fetal DNA Concentrations in Women Who Eventually Develop Preeclampsia.Clin Chem. 2001; 47: 137-139PubMed Google Scholar, 7Zhong XY Holzgreve W Hahn S The Levels of Circulatory Cell Free Fetal DNA in Maternal Plasma Are Elevated Prior to the Onset of Preeclampsia.Hypertens Pregnancy. 2002; 21: 77-83Crossref PubMed Scopus (150) Google Scholar and monitor8Swinkels DW de Kok JB Hendriks JC Wiegerinck E Zusterzeel PL Steegers EA Hemolysis. Elevated Liver Enzymes, and Low Platelet Count (Hellp) Syndrome as a Complication of Preeclampsia in Pregnant Women Increases the Amount of Cell-Free Fetal and Maternal DNA in Maternal Plasma and Serum.Clin Chem. 2002; 48: 650-653PubMed Google Scholar the severity of complicated pregnancies. Yet, the low percentage of fetal cfDNA (3% to 6%) in maternal plasma limits its use for routine clinical applications. As a result, many groups, including ours, have focused on methods to enhance recovery and analysis of circulating fetal cfDNA. To achieve this goal, a better understanding of the molecular nature and kinetics of circulating (fetal) DNA is necessary. We hypothesize that circulating DNA could exist as free DNA subject to degradation as well as DNA contained in membrane bound MPs, providing protection from endonucleases. In 2005, we proposed that a significant portion of circulating DNA, including fetal DNA, was membrane bound and suitable for enrichment.9Bischoff FZ Lewis DE Simpson JL Cell-Free Fetal DNA in Maternal Blood: kinetics.Source and Structure Hum Reprod Update. 2005; 11: 59-67Crossref PubMed Scopus (137) Google Scholar We reported a 10-fold enrichment of fetal cfDNA from maternal plasma from normal pregnancies using the flow cytometric parameters of forward light scatter and acridine orange (AO) staining of nucleic acids. Although the nature and origin of the AO+ DNA was unknown, we suggested that the mechanism of release of fetal cfDNA might be due to placental apoptosis resulting in release of fetal DNA containing MPs into the circulation. The plausibility of this hypothesis was supported by data concerning turnover of syncytiotrophoblast and release of syncytiotrophoblast MPs (STBMs) into the maternal circulation.10Huppertz B Frank HG Kingdom JC Reister F Kaufmann P Villous Cytotrophoblast Regulation of the Syncytial Apoptotic Cascade in the Human Placenta.Histochem Cell Biol. 1998; 110: 495-508Crossref PubMed Scopus (358) Google Scholar In addition, Gupta et al detected fetal nucleic acids in the supernatant of STBM samples prepared from placental explants.11Gupta AK Holzgreve W Huppertz B Malek A Schneider H Hahn S Detection of Fetal DNA and Rna in Placenta-Derived Syncytiotrophoblast Microparticles Generated in Vitro.Clin Chem. 2004; 50: 2187-2190Crossref PubMed Scopus (87) Google Scholar Other studies have also examined STBMs from preeclamptic patients using an enzyme-linked immunosorbent assay based method for quantification.12Knight M Redman CW Linton EA Sargent IL Shedding of Syncytiotrophoblast Microvilli into the Maternal Circulation in Pre-Eclamptic Pregnancies.Br J Obstet Gynaecol. 1998; 105: 632-640Crossref PubMed Scopus (409) Google Scholar, 13Goswami D Tannetta DS Magee LA Fuchisawa A Redman CW Sargent IL von Dadelszen P Excess Syncytiotrophoblast Microparticle Shedding Is a Feature of Early-Onset Pre-Eclampsia, but Not Normotensive Intrauterine Growth Restriction.Placenta. 2006; 27: 56-61Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 14Germain SJ Sacks GP Sooranna SR Sargent IL Redman CW Systemic Inflammatory Priming in Normal Pregnancy and Preeclampsia: the Role of Circulating Syncytiotrophoblast Microparticles.J Immunol. 2007; 178: 5949-5956PubMed Google Scholar Whether the STBMs contain nucleic acids similar to the AO+ MPs sorted from maternal plasma has not been studied. One of the biochemical hallmarks of apoptosis is fragmented DNA. DNA is cleaved between nucleosomes, forming a "ladder" with spacing about 180 bp. Given that the majority of fetal cfDNA is relatively small (<300 bp, as detected by PCR) compared to maternal cfDNA,15Chan KC Zhang J Hui AB Wong N Lau TK Leung TN Lo KW Huang DW Lo YM Size Distributions of Maternal and Fetal DNA in Maternal Plasma.Clin Chem. 2004; 50: 88-92Crossref PubMed Scopus (461) Google Scholar fetal DNA is likely released from apoptotic cells. However, the basis for fetal DNA stability has not been examined. More recently, Tjoa et al demonstrated an association of fetal cfDNA (as detected by β-globin via real-time PCR) and cell death in supernatants from third trimester placental cultures after oxidative stress,16Tjoa ML Cindrova-Davies T Spasic-Boskovic O Bianchi DW Burton GJ Trophoblastic Oxidative Stress and the Release of Cell-Free Feto-Placental DNA.Am J Pathol. 2006; 169: 400-404Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar supporting the hypothesis that fetal cfDNA originates from the placenta via hypoxia-induced cell death, apoptosis and necrosis. However, the DNA detected was not examined as to source or characteristics. Recovery of trophoblast MPs containing fetal DNA during the first trimester would be of clinical interest to prenatal genetic diagnosis. However, under hypoxic conditions, few syncytiotrophoblasts are detected in first trimester placental explants17Huppertz B Kingdom J Caniggia I Desoye G Black S Korr H Kaufmann P Hypoxia Favours Necrotic Versus Apoptotic Shedding of Placental Syncytiotrophoblast into the Maternal Circulation.Placenta. 2003; 24: 181-190Abstract Full Text PDF PubMed Scopus (253) Google Scholar and circulating STBMs are not found until the second and third trimester.13Goswami D Tannetta DS Magee LA Fuchisawa A Redman CW Sargent IL von Dadelszen P Excess Syncytiotrophoblast Microparticle Shedding Is a Feature of Early-Onset Pre-Eclampsia, but Not Normotensive Intrauterine Growth Restriction.Placenta. 2006; 27: 56-61Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar By contrast, invasive extravillous trophoblasts (EVTs) are most abundant during the first trimester.18Kovats S Main EK Librach C Stubblebine M Fisher SJ DeMars R A Class I Antigen. Hla-G, Expressed in Human Trophoblasts.Science. 1990; 248: 220-223Crossref PubMed Scopus (1229) Google Scholar In addition, apoptosis limits invading EVTs during the first trimester,19Reister F Frank HG Kingdom JC Heyl W Kaufmann P Rath W Huppertz B Macrophage-Induced Apoptosis Limits Endovascular Trophoblast Invasion in the Uterine Wall of Preeclamptic Women.Lab Invest. 2001; 81: 1143-1152Crossref PubMed Scopus (250) Google Scholar suggesting that MPs released by dying EVTs might enter the maternal circulation in the first trimester. Primary trophoblasts isolated from villous explant cultures contain a heterogeneous population of cytotrophoblasts, syncytiotrophoblasts, fibroblasts, and macrophages.20James JL Stone PR Chamley LW The Isolation and Characterization of a Population of Extravillous Trophoblast Progenitors from First Trimester Human Placenta.Hum Reprod. 2007; 22: 2111-2119Crossref PubMed Scopus (48) Google Scholar, 21Chu W Fant ME Geraghty DE Hunt JS Soluble Hla-G in Human Placentas: synthesis in Trophoblasts and Interferon-Gamma-Activated Macrophages but Not Placental Fibroblasts.Hum Immunol. 1998; 59: 435-442Crossref PubMed Scopus (76) Google Scholar JEG-3 cells, which model invasive EVTs, most relevant for early genetic testing, have the advantage of being homogeneous with less spontaneous cell death than found with primary trophoblasts.22Al-Nasiry S Spitz B Hanssens M Luyten C Pijnenborg R Differential Effects of Inducers of Syncytialization and Apoptosis on Bewo and Jeg-3 Choriocarcinoma Cells.Hum Reprod. 2006; 21: 193-201Crossref PubMed Scopus (77) Google Scholar Therefore, we used JEG-3 cells to represent EVTs. Our studies show that cfDNA is contained within membranous MPs derived from dying JEG-3 cells. We also showed for the first time that the DNA within the apoptotic MPs contain fragmented DNA that is protected from plasma DNases. In addition, light scatter and DNA staining properties of in vitro cell derived MPs and maternal plasma MPs are similar, suggesting a common origin or mechanism of release. We also show that in vitro MPs and MPs from normal and preeclamptic pregnancies stain with the DNA dye, PicoGreen. Finally, we show for the first time that significantly higher numbers of DNA+ MPs are found in preeclamptic patients compared to control patients, suggesting that measuring such DNA+ MPs could help predict or monitor preeclampsia in a non-invasive manner. Other clinical and pathological conditions associated with increased levels of cell death (ie, trauma, stroke, organ transplantation, autoimmunity, and cancer) might also benefit from our discovery of circulating nucleic acids containing protected MPs. The human trophoblastic cell line, JEG-3 (male), was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in minimum essential medium, supplemented with heat inactivated 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mmol/L l-glutamine, 1 mmol/L sodium pyruvate, 10 mmol/L HEPES, and 0.1 mmol/L non-essential amino acids (Invitrogen, Carlsbad, CA). The cultures were maintained at 37°C in an atmosphere of 5% CO2. Two million cells were cultured in the presence or absence of either 50 μmol/L rotenone, which disrupts the mitochondrial electron transport chain to chemically mimic hypoxia (Sigma-Aldrich, St Louis, MO), or 30 μmol/L etoposide, a DNA topoisomerase II inhibitor (Sigma-Aldrich) that induces apoptosis. For necrosis, cells were heated for 1 hour at 60°C. Twenty-four and 48 hours after induction of cell death, floating cells were collected and adherent cells were detached with trypsin/EDTA (Invitrogen). The cells were pooled, centrifuged at 300 × g at room temperature, rinsed in PBS (pH 7.1; Invitrogen) and centrifuged at 300 × g. Apoptotic cell death was confirmed by flow cytometric analysis of fixed (permeabilized) cells with a subdiploid DNA content.23Alonso MM Asumendi A Villar J Gil MJ Martinez-Merino V Encio IJ Migliaccio M New Benzo(B)Thiophenesulphonamide 1,1-Dioxide Derivatives Induce a Reactive Oxygen Species-Mediated Process of Apoptosis in Tumour Cells.Oncogene. 2003; 22: 3759-3769Crossref PubMed Scopus (25) Google Scholar While gently vortexing, 1 × 106 cells (counted with a hemocytometer) were resuspended in 2 ml of 0.9% NaCl, followed by fixation with 5 ml ice cold 70% ethanol added dropwise. Cells were incubated for 1 hour at room temperature, centrifuged, and resuspended in 1 ml PBS containing 50 μg/ml propidium iodide (PI) (Sigma-Aldrich) and 0.1 mg/ml RNase A (Sigma-Aldrich), incubated for 1 hour at 37°C in the dark. Cells were analyzed using a Beckman Coulter EPICS XL2 (Beckman Coulter, Miami Lakes, FL). All data analysis was performed using EXPO32 software (Beckman Coulter). PI was used to test for membrane integrity using unfixed cells as previously described.23Alonso MM Asumendi A Villar J Gil MJ Martinez-Merino V Encio IJ Migliaccio M New Benzo(B)Thiophenesulphonamide 1,1-Dioxide Derivatives Induce a Reactive Oxygen Species-Mediated Process of Apoptosis in Tumour Cells.Oncogene. 2003; 22: 3759-3769Crossref PubMed Scopus (25) Google Scholar One million (nonpermeabilized) cells were resuspended in PBS containing 50 μg/ml PI and analyzed by flow cytometry. Isolation and quantification of MPs are performed before all experiments. Double filtered (0.25 μm) PBS (dfPBS) was used in all MP experiments. MPs were centrifuged at room temperature, unless otherwise stated. MPs from 24 and 48 hours apoptotic and necrotic supernatants were separated from detached cells by two centrifugation steps (300 × g, 5 minutes; 800 × g, 5 minutes) and transferred to a 50 ml conical tube. MP concentration was determined as previously described.24Montes M Jaensson EA Orozco AF Lewis DE Corry DB A General Method for Bead-Enhanced Quantitation by Flow Cytometry.J Immunol Methods. 2006; 317: 45-55Crossref PubMed Scopus (42) Google Scholar Briefly, 100 μl of fluorescent beads (counted at 500/μl with a hemocytometer) were added to 400 μl of each supernatant and 300 μl of dfPBS for a total volume of 800 μl and the number of beads counted was stopped at 10,000. After subtracting the number of background fluorescent beads, the concentration of MPs was determined and the appropriate number of MPs was centrifuged (25,000 × g, 1 hour at 4°C; in a SW40 Ti Swinging-Bucket Rotor in a Beckman L8-M, Class H, ultracentrifuge; Beckman Coulter). Because lipid membrane MPs protect cell-free RNA (cfRNA) from RNase A activity (Table 1), we found it necessary to fix the MPs with 70% ethanol to permeabilize the membranes, before RNase treatment. Two million MPs were fixed with 70% ethanol (as described above) and centrifuged (13,000 rpm, 10 minutes) using an Eppendorf Centrifuge 5415c (Eppendorf, Westbury, NY). The MP pellets were resuspended in 500 μl PBS containing 10 μg/ml of Hoechst 33342 (Invitrogen), with or without 50 μl of 1 mg/ml RNase A. After incubating the samples for 20 minutes at 37°C in a water bath, the samples were placed on ice and analyzed using an LSR II flow cytometer (BD Instruments, San Jose, CA).Table 1RNA from Apoptotic MPs Is Protected by Triton X-100 Extractable LipidsSampleTreatmentsCt ValueA-MPUntreated22A-MP200 μg/ml RNase21A-MPTx-100 + 200 μg/ml RNase40A-MP1000 μg/ml RNase19A-MPTx-100 + 1000 μg/ml RNase40A-MP, apoptotic MPs.Tx-100, Triton X-100.n = 1. Open table in a new tab A-MP, apoptotic MPs. Tx-100, Triton X-100. n = 1. MPs (2 × 106) in 50 μl were placed on a glass slide (Superfrost Plus Micro Slides; VWR Scientific, West Chester, PA) and air-dried at room temperature. The slides were fixed in a Coplin jar containing 25 ml of 100% methanol for 10 minutes and allowed to air-dry. The slides were immersed in a second Coplin jar containing 25 ml of 2× standard saline citrate with 0.2% NP40 for 1 minute and the edges were dried with a tissue. A final concentration of 4,6-diamidino-2-phenylindole (DAPI II, 1 μg/ml, Invitrogen) was placed on the surface of a glass coverslip, inverted onto a slide and analyzed by fluorescence microscopy. Images of the DAPI-stained MPs were captured through an Olympus BX51 microscope with automated x-, y-, and z-stage movement and a Hamamatsu ORCA-2 Digital Camera using a ×100 objective (Hamamatsu Photonics, Hamamatsu, Japan). Data were analyzed using Advanced Digital Imaging Research automated interphase FISH Scanner software (Advanced Digital Imaging Research L.L.C., League City, TX). Each slide was autoscanned for single MPs ranging 1 μm to 6 μm in diameter to avoid clustering of MPs or possible cell contamination. An average of 3000 to 8000 images was taken per slide. Electronic gates (boxes 1 μm to 6 μm in length) were placed around each MP from randomly selected images and the integrated fluorescence intensity was determined. One hundred MPs per slide, per experiment were counted. The amount of DNA per particle was reported as the mean integrated fluorescence intensity (MIFI). DNA was extracted from 1 to 2 million MPs using the QIAamp DNA blood kit (Qiagen, Valencia, CA) according to manufacturer's instructions. All DNA samples were stored at 4°C before analysis. Real-time PCR was performed as previously described.25Jorgez CJ Dang DD Simpson JL Lewis DE Bischoff FZ Quantity Versus Quality: optimal Methods for Cell-Free DNA Isolation from Plasma of Pregnant Women.Genet Med. 2006; 8: 615-619Crossref PubMed Scopus (49) Google Scholar For both the β-globin (102 bp) and sex-determining region Y (SRY, 72 bp), quantitative real-time (RT)-PCR was performed using the Applied Biosystems 7700 sequence detection system (Applied Biosystems, Foster City, CA). Primer and probe sequences were as follows: SRY forward primer: 5′:-TGCACAGAGAGAAATACCCGAATTA-3′; SRY reverse primer: 5′:-TGCATTCTTCGGCAGCAT-3′; SRY TaqMan probe: 5′:-AAGTATCGACCTCGTCGGAAGGCGAA-3′; β-globin forward primer: 5′:-GTGCACCTGACTCCTGAGGAGA-3′; β-globin reverse primer: 5′:-CCTTGATACCAACCTGCCCAG-3′; β-globin TaqMan probe: 5′:-AAGGTGAACGTGGATGAAGTTGGTGG-3′. Quantification of total and fetal DNA as genome equivalents was based on copies of β-globin and SRY sequences detected. Each reaction contained 5 μl of extracted DNA. Each reaction plate was run simultaneously with a duplicate calibration curve of titrated DNA (standard curve). Each sample was run in triplicate for both loci and the mean of the values was determined using the 7700 software and the standard curve of known DNA concentrations. To determine the relative amount of DNA per MP, the total (β-globin) and fetal DNA (SRY) genome equivalents were divided by the number of MPs. DNA was extracted from fifteen million MPs and analyzed on a 2% agarose gel with a 1-kb DNA ladder (Invitrogen). MPs were resuspended in 300 μl of lysis buffer (10 mmol/L Tris-HCl-EDTA) with 50 μg/ml RNase A for 1 hour at 37°C. MPs were incubated overnight (∼12 hours) at 37°C in 0.05% SDS with 2 mg/ml of Proteinase K (Sigma-Aldrich). An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1; Invitrogen) was added and the tubes were gently inverted twenty times. The samples were centrifuged (13,000 rpm, 10 minutes) and the top layer (aqueous) was removed and transferred to second tube. An equal volume of chloroform (99+%; Sigma-Aldrich) was added and the samples were centrifuged (13,000 rpm, 10 minutes). The top layer (aqueous phase) was removed and transferred to a third tube. DNA was precipitated by adding one-third volume of 7.5 mol/L ammonium acetate (Sigma-Aldrich), followed by 2.5 volumes of absolute ethanol, and precipitated at −80°C for 30 minutes. The precipitates were collected by centrifugation (13,000 rpm, 10 minutes) and the pellets were washed with 70% ethanol and air-dried. The proteinase K, phenol/chloroform/isoamyl alcohol (25:24:1), and DNA precipitation steps were repeated a second time. The final DNA pellet was air-dried and resuspended in 60 μl of Tris-HCl-EDTA (10 mmol/L) and DNA concentration determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Three micrograms of DNA were electrophoresed on a 2% agarose gel (stained with 0.2 μg/ml ethidium bromide) and analyzed with Kodak Image Station 4000 mm Pro (Eastman Kodak Company, Molecular Imaging Systems, Rochester, NY). DNA nicks were detected by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay as previously described26Darzynkiewicz Z Galkowski D Zhao H Analysis of Apoptosis by Cytometry Using Tunel Assay.Methods. 2008; 44: 250-254Crossref PubMed Scopus (167) Google Scholar with slight modifications. Analysis was by flow cytometry and fluorescence microscopy with the following buffers: 5× TdT reaction buffer (1 mol/L potassium cacodylate [Sigma-Aldrich], 125 mmol/L Tris-Cl, pH 6.6, and 1.25 mg/ml bovine serum albumin [Sigma-Aldrich] stored at −20°C); and fluorescein isothiocyanate-conjugated anti-bromolated deoxyuridine triphosphates (Br-dUTP) monoclonal antibody solution (Anti-Br-dUTP fluorescein isothiocyanate-conjugated monoclonal antibody, 0.5 μg, Phoenix Flow Systems, San Diego, CA; 0.3% (v/v) Triton X-100; 1% (w/v) bovine serum albumin (Sigma-Aldrich); and PBS to 100 μl (stored at 4°C). Briefly, two million MPs from chemically-induced hypoxic cells and heat stress (HS)-treated cells (at 60°C) were pre-fixed in 1 ml of 2% methanol-free formaldehyde (Electron Microscopy Sciences, Hatfield, PA) diluted in PBS on ice for 1 hour. The MPs were centrifuged (25,000 × g, 1 hour at 4°C) and postfixed in 1 ml of ice-cold 70% ethanol for 5 hours at −20°C. MPs were centrifuged (13,000 RPM, 10 minutes), the ethanol removed, and the MPs washed in 1 ml of PBS. MPs were resuspended in 50 μl of labeling buffer (10 μl 5× TdT reaction buffer, 2 μl Br-dUTP stock solution [2 mmol/L Br-dUTP; Sigma-Aldrich], 0.5 μl [12.5U] TdT enzyme [Invitrogen], 5 μl CoCl2 [10 mmol/L; Sigma-Aldrich] and 33 μl distilled water) and incubated at room temperature overnight. Three hundred microliters of rinsing buffer (0.1% [v/v] Triton X-100, 5 mg/ml BSA, and PBS, pH 7.4) was added and the MPs centrifuged (13,000 RPM, 10 minutes). The supernatant was removed and 100 μl fluorescein isothiocyanate-conjugated anti-Br-dUTP monoclonal antibody was added to the MPs and incubated for 1 hour at room temperature. Four hundred microliters of PBS-based PI staining solution (50 μl of 1 mg/ml PI [Sigma-Aldrich] and 50 μl of 1 mg/ml RNase A [Sigma-Aldrich]) was added and incubated for 30 minutes at room temperature in the dark. The samples were analyzed by a Beckman Coulter EPICS XL2. After labeling MPs with Br-dUTP incubated at room temperature overnight, a 50 μl aliquot of two million apoptotic or necrotic MPs was air-dried onto slides. MPs were counterstained with DAPI II (Invitrogen, as described above). The double labeled MPs were analyzed with an Olympus BX-51 Microscope and photographed with a Hamamatsu ORCA-2 Digital Camera using an ×100 objective (Hamamatsu, Photonics). Scale bars are 10 μm. Two million MPs from each sample were labeled with PKH26 Red Fluorescent Cell Linker Kit (Sigma) as described by the manufacturer with slight modifications. MPs were washed once with PBS (Invitrogen) and stained with 100 μl of staining solution (5 × 10−5 mol/L PKH26 in diluent C) for 4 minutes at room temperature. The staining reaction was stopped by addition of 100 μl of fetal calf serum. One minute later, 100 μl of complete medium were added and MPs were washed twice in 10% minimum Essential medium, then once in PBS. MPs were resuspended in 500 μl PBS containing 10 μg/ml of Hoechst 33342 (Invitrogen), incubated for 20 minutes in a water bath at 37°C, placed on ice, and analyzed using an LSR II flow cytometer. Two million MPs resuspended in 500 μl PBS were stained with 5 μl of cholera toxin B (CTB, 1 mg/ml, Invitrogen) and incubated on ice for 10 minutes. MPs were labeled with 4 μg/ml of Hoechst 33342 (Invitrogen), prepared as described on above, and analyzed using an LSR II flow cytometer. After obtaining Institutional Review Board approval from Baylor College of Medicine and written informed consent, 10 ml of peripheral blood was collected from a non-pregnant female in a syringe containing 10 μl/ml of anticoagulant heparin sulfate (5000 U/ml) and allowed to sit at room temperature for 1 hour. Blood was centrifuged for 10 minutes at 800 × g (room temperature), followed by a second centrifugation at 1600 × g. The cell-free plasma was used to incubate the in vitro MPs as described below. Five million MPs from each sample were incubated in 800 μl of the non-pregnant female plasma for 24 hours on ice and in a 37°C water bath. DNA was extracted using the MagNA Pure LC System (Roche Diagnostics) according to manufacturer's instructions. Purified male gDNA (Promega, Madison, WI) and a water blank were used to create a standard curve (1.5 ng to 245 ng DNA) to determine the linearity and range of the PCR products. PCR was performed using primers specific to a 193 bp SRY PCR product, using the following primers: SRY forward primer: 5′:-AAAGGCAACGTCCAGGATAGAG-3′; SRY reverse primer: 5′:-TGTAATTTCTGTGCCTCCTGGA-3′. Controls used in all tests included a male positive control for SRY and no DNA (water) control. The PCR reaction (55 μl) included 5 μl 1× PCR buffer, 2 μl 50 mmol/L MgCl, 1.5 μl 100 mmol/L dNTPs, 2.5 μl 100 mmol/L SRY-Forward, 2.5 μl 100 mmol/L SRY-Reverse, 15 μl (30 ng) DNA, 0.3 μl (500 U) Taq polymerase, and 26.2 μl water (Invitrogen). PCR amplification was performed using a hot start method (10 minutes at 95°C.), followed by 35 cycles of denaturation at 94°C for 1 minute, re-annealing at 63.2°C for 1 minute, and extension at 72°C for 1 minute, and one final extension of 10 minutes at 72°C. The PCR products were run on a 1% agarose gel, stained with 0.2 μg/ml ethidium bromide, and the band intensities were scanned using the Kodak Digital Science Image Station 440CF (Eastman Kodak Company, Molecular Imaging Systems). Band intensities were determined using the Image J version 1.37v software (developed at the National Institutes of Health). A standard curve was generat
Referência(s)