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Cell totally free supernatants were analysed for IFN- and IL-4 protein and so are expressed simply because picogram (pg)/106 cells

Cell totally free supernatants were analysed for IFN- and IL-4 protein and so are expressed simply because picogram (pg)/106 cells. Fluorescence-activated cell sorting (FACS) tests showed that IFN- mRNA was upregulated by IL-15 in both Compact disc4+ and Compact disc8+ T lymphocytes, whereas IL-4 mRNA deposition occurred in Compact disc4+ cells. Preincubation of extremely purified Compact disc4+ T lymphocytes during seven days with IL-15 and/or IL-7, accompanied by activation, demonstrated improved IL-4 proteins secretion Fli1 also, but upregulated IFN- protein mostly. The web effect was a increased IFN-/IL-4 ratio. Taken jointly, IL-15 and IL-7 can become costimulatory signals, which might favour a T helper 1 (Th1) immune system response, in the lack of sufficient CD28 costimulation particularly. Launch Interleukin (IL)-15, a identified cytokine recently, is normally made by a multitude of tissue and cells, such as 4-Aminohippuric Acid for example epithelial and monocytic cells in response to endotoxin arousal.1,2 The consequences of IL-15 are focus on reliant. IL-15 enhances different monocytic features, boosts interferon- (IFN-) creation by organic killer cells and it is a chemoattractant for T lymphocytes.3,4 Recently, it’s been demonstrated that IL-15 promotes the creation from the T helper 2 (Th2)-like cytokine IL-5 by individual helper T lymphocytes.5 The IL-15-mediated effects are transmitted 4-Aminohippuric Acid with the IL-15 receptor which consists partly from the -and C chain from the IL-2 receptor.6 IL-7 is a cytokine which affects T lymphocyte functions profoundly. 7C11 Although IL-7 enhances the creation of cytokines such as for example IL-4 and IL-3,12,13 it preferentially stimulates the Th1-type mediated immune response by improving the production of IL-2 and IFN-14.15 The observed ramifications of IL-7 are mediated by the precise IL-7 receptor chain with the IL-2 receptor C chain.16,17 Thus, differential ramifications of IL-7 and IL-15 on Th1-and Th2-type cytokine appearance are mediated partially with the shared IL-2R C string. Therefore, it could be of interest to review whether differences can be found in effector features of IL-15 and IL-7 on T lymphocytes, specifically regarding the legislation of Th1- and Th2-type cytokines. In today’s study we’ve analysed the consequences of IL-15 and IL-7 over the gene appearance of IFN- (Th1-type) and IL-4 (Th2-type) in turned on individual T lymphocytes. Although IL-7 and IL-15 improved IL-4 gene appearance, the upregulatory aftereffect of IL-7 and IL-15 on IFN- was most pronounced. The web result being truly a increased IFN-/IL-4 ratio. We conclude that IL-15 and IL-7 may become costimulatory indicators for T lymphocyte activation and could favour a Th1-type response. Strategies and Components Planning of cellsPeripheral bloodstream cells had been extracted from healthful volunteer platelet donors, and mononuclear cell suspensions had been made by FicollCHypaque (Lymphoprep, Nycomed, Oslo, Norway) density-gradient centrifugation as defined before.12C14,18 T lymphocytes were isolated by 2-aminoethylisothiouronium bromide (AET) treated sheep red bloodstream cell (SRBC) rosetting. The SRBC had been lysed with 155 mmol/l NH4Cl, 10 mmol/l KHCO3, 01 mmol/l ethylenediamine tetra-acetic acidity (EDTA) and the rest of the cell preparations included a lot more than 98% T lymphocytes as evaluated by stream cytometric evaluation after staining with an anti-CD2 monoclonal antibody (Becton Dickinson, Hill Watch, CA) and significantly less than 1% Compact disc14-positive cells (Becton Dickinson). After isolation, T lymphocytes had been cultured right away at 37 in RPMI-1640 mass media (Stream, Rockville, MD) filled with 4% fetal bovine serum (FBS; Hyclone, Logan, UT) supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 2 mmol/l l-glutamine and 6 ng/ml colistine. Compact disc4+ and Compact disc8+ T lymphocytes had been attained with a fluorescence cell sorting technique on the FACS-Star stream cytometer (Becton Dickinson) using fluoroscein isothiocyanate (FITC)-labelled Compact disc4-and phycoerythrin-labelled Compact disc8-antibodies (Becton Dickinson). Purity from the attained Compact disc4+ and Compact disc8+ subpopulations was 99% as evaluated by re-analysis. These extremely purified Compact disc4+ T lymphocytes included 10% Compact disc56+ cells. Appearance of Compact disc25 and Compact disc56 was performed by FACS-Star stream cytometry using FITC-labelled Compact disc25 (Becton Dickinson) and Compact disc56 (Becton Dickinson) antibodies. StimulationT cells (5106/ml) from five different donors had been activated with 25 g/ml concanavalin A (Con A; Calbiochem, La Jolla, CA) or by an optimum focus of anti-CD3 and anti-CD28 antibodies within a 5% hybridoma supernatant alternative14,18 (present from Dr B. J. Kroesen, Dept of Immunology, School of Groningen, HOLLAND). For the priming tests purified Compact disc4+ T lymphocytes (106/ml) had been preincubated with moderate (RPMI-1640), IL-15, IL-7 or mix of IL-15 plus IL-7 utilized at optimal concentrations.14 After seven days of incubation the cells were washed twice and put into fresh moderate (RPMI-1640). The viability from the cells was 98% as dependant on trypan blue exclusion. After keeping 4-Aminohippuric Acid track of the cells, 106 cells had been turned on with Con An advantage phorbol myristate acetate (PMA; Sigma, St. Louis, MO) to maximally induce cytokine secretion. PMA was utilized at an optimum concentrations of.

When considering which of the numerous objective measures of adherence to make use of, it’s important to consider the individual obstacles and people to make use of and/or data collection

When considering which of the numerous objective measures of adherence to make use of, it’s important to consider the individual obstacles and people to make use of and/or data collection. Following randomization as well as the baseline assessment, females are given using the involvement components and so are contacted with a scholarly research nurse to begin with involvement periods. process will be examined seeing that extra final results. Individuals (N=400) will end up being recruited from a tertiary treatment infirmary or community treatment centers in clinically underserved or rural areas. Individuals will end up being randomized to get CST-AET or an over-all health education involvement (evaluation condition). CST-AET contains ten nurse-delivered phone calls delivered over six months. CST-AET provides organized trained in coping abilities for handling symptoms that hinder adherence. Interactive tone of voice messaging provides support for abilities make use of and adherence that’s tailored predicated on real-time adherence data Doxapram in the wireless smart tablet bottles. Provided the high prices of non-adherence and latest recommendations that ladies stick to AET for a decade, Doxapram we explain a timely trial. If effective, the CST-AET process may not just decrease the burden of AET make use of but also result in cost-effective adjustments in clinical treatment and improve breasts cancer outcomes. is normally to research the impact from the CST-AET process on adherence to AET. Our is normally to examine the influence from the the CST -AET involvement process on symptom disturbance. Our is normally to examine the influence from the CST -AET process on theory-based methods of perceived obstacles to AET medicine, values about AET medicine, and self-efficacy. Finally, our is normally to estimation short-term costs of applying the CST-AET involvement and investigate long-term cost-effectiveness from the CST-AET involvement. B. Individual Selection a. Eligibility Doxapram Requirements Clinical trial individuals are recruited from a tertiary infirmary and through community cancers clinics associated with a cancers clinic network from the tertiary infirmary. These community cancers clinics can be found in underserved or rural communities medically. Eligible participants meet up with the pursuing inclusion requirements: a) medical diagnosis of Stage I to III breasts cancer tumor, b) hormone receptor positive tumor thought as any positivity of estrogen or progesterone receptor, c) finished procedure, chemotherapy, and rays, d) within a year of starting AET, and e) at least 1 . 5 years of AET suggested. Exclusion requirements are: a) 21 years, b) serious cognitive or hearing impairment that’s noted in the medical record, or c) struggling to offer significant consent (e.g., serious cognitive impairment). Though regarded, we chose never to consist of eligibility criteria linked to the particular level or kind of symptoms females are suffering from as symptoms can fluctuate and transformation during the period of AET and could differ by kind of AET treatment.57-59 b. Subject matter Recruitment This scholarly research was accepted by the Institutional Review Plank. Recruitment techniques with HIPAA suggestions comply. Patients conference eligibility criteria are given information about the analysis in another Doxapram of two methods: 1) the analysis brochure that briefly represents the analysis and letter explaining the study is normally given to females by an associate of their treatment group during an oncology follow-up go to, or 2) the analysis brochure and a notice off their doctor presenting the study is normally mailed to them. Potential individuals are telephoned by research personnel and asked if they’re thinking about hearing about the analysis. For girls who express curiosity, research personnel arrange an in-person conference to help expand describe the scholarly research, confirm eligibility, and acquire up to date consent. C. Techniques The scholarly research style and timeline are presented in amount 1. Because of this RCT, 400 females who are recommended AET for breasts cancer tumor are randomized with identical allocation to 1 of two groupings: 1) a coping abilities training involvement for enhancing abilities to boost adherence and reduce indicator interference (CST-AET; energetic involvement group), or 2) health and wellness education (evaluation group that handles for period and attention). Randomization depends upon a randomization plan. Participants comprehensive assessments within their dealing with medical clinic at baseline, three months (at bottom line of involvement sessions), six months (at bottom line of maintenance calls), 12 months, and 18 Rabbit Polyclonal to KCNK15 months (one year after conclusion of maintenance calls). A set of questionnaires takes approximately 45-60 minutes to complete and objective physical assessments (i.e., six minute walk test, timed get up and go test, and grip strength test) take approximately 14-20 minutes to complete. Whenever possible, assessments are completed in-person so that participants can complete the physical assessments and research.

Although abundantly expressed in the nucleolus, Nucleolin also localizes to cytoplasmic and plasma membrane compartments [37C39], and its subcellular localization can indicate its functional role in different cell types [35]

Although abundantly expressed in the nucleolus, Nucleolin also localizes to cytoplasmic and plasma membrane compartments [37C39], and its subcellular localization can indicate its functional role in different cell types [35]. to siCONT cells. Transfected HeLa cells were also assessed for changes in (C) migration and (D) invasion 6 h or 48 h following introduction of chemoattractant (FBS), respectively. siPVT1 cells showed a significant decrease in both cell migration and invasion compared to siCONT cells. Quantitative results are graphed on the left, while representative images are on the right. SC75741 **p<0.01, ***p<0.001, ****p<0.0001(TIF) pone.0156274.s003.tif (5.1M) GUID:?15E8C744-A6E3-479D-801B-3D52862BF6DE S4 Fig: PVT1 binds, but does not stabilize, Myc in SiHa cells. (A) PVT1 from SiHa total cell lysate immunoprecipitated with a MYC antibody, but not the negative control rabbit IgG (Cont). (B) PVT1 knockdown in SiHa cells did not significantly affect p58 phospho-MYC or total MYC protein. (C,D) Degradation of MYC protein was also not significantly affected by PVT1 knockdown. **p<0.01(TIF) pone.0156274.s004.tif (476K) GUID:?70EA7899-A6B4-4EAC-AC40-509BCE69A1CE S5 Fig: knockdown does not affect Nucleolin protein levels. (TIF) pone.0156274.s005.tif (971K) GUID:?84FAEDA5-E4E5-4D26-850B-5741EB99BB71 S1 Table: Percent knockdown of different regions achieved with siRNA or LNA. * denotes primer set used throughout the manuscript.(XLSX) pone.0156274.s006.xlsx (10K) GUID:?1FA64BD0-D3A8-45F1-A18E-BCA497DA3102 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract The plasmacytoma variant translocation 1 gene (influences disease processes has been studied in multiple cancer types, its role in cervical tumorigenesis remains unknown. Thus, the present study was designed to investigate the role of in cervical cancer and expression was measured by quantitative PCR (qPCR) in 121 invasive cervical carcinoma (ICC) samples, 30 normal cervix samples, and cervical cell lines. Functional assays were carried out using both siRNA and LNA-mediated knockdown to examine expression is SC75741 significantly increased in ICC tissue versus normal cervix and that higher expression of correlates with poorer overall survival. In cervical cancer cell lines, knockdown resulted in significantly decreased cell proliferation, migration and invasion, while apoptosis and cisplatin cytotoxicity were significantly increased in these cells. Finally, we show that expression is augmented in response to hypoxia and immune response stimulation and that this lncRNA associates with the multifunctional and stress-responsive protein, Nucleolin. Collectively, our results SC75741 provide strong evidence for an oncogenic role of in cervical cancer and lend insight into potential mechanisms by which overexpression helps drive cervical carcinogenesis. Introduction Long noncoding RNAs (lncRNAs) provide important targets for cancer diagnostics and therapeutics due to their critical role in numerous cellular processes such as epigenetic changes, gene enhancer and tumor suppressor activity, and miRNA sequestering. LncRNAs are pervasive in the genome, frequently show cell type- and temporal-specific regulation of gene expression, and can influence Mouse monoclonal antibody to AMPK alpha 1. The protein encoded by this gene belongs to the ser/thr protein kinase family. It is the catalyticsubunit of the 5-prime-AMP-activated protein kinase (AMPK). AMPK is a cellular energy sensorconserved in all eukaryotic cells. The kinase activity of AMPK is activated by the stimuli thatincrease the cellular AMP/ATP ratio. AMPK regulates the activities of a number of key metabolicenzymes through phosphorylation. It protects cells from stresses that cause ATP depletion byswitching off ATP-consuming biosynthetic pathways. Alternatively spliced transcript variantsencoding distinct isoforms have been observed many cellular processes via multiple disparate mechanisms [1]. Plasmacytoma variant translocation 1 (that has attracted significant attention from the cancer field due to its frequent co-amplification with in several solid tumors [2]. The first studies providing evidence that may contribute to carcinogenesis demonstrated frequent translocations in mouse plasmacytomas [3,4] and human Burkitts lymphomas [5C7]. The oncogenic effects of have been further highlighted by more recent studies demonstrating its overexpression and amplification in multiple cancer types [8C17]. More importantly, expression has been significantly correlated with clinical features such as risk, recurrence, and survival in various cancers [8,11C13,18]. Despite the wealth of knowledge regarding the oncogenic properties of in multiple cancers, very little is known about its precise biologic function. In fact, the handful of studies providing mechanistic data exceedingly suggest that it exerts its effects in a cell-type and/or disease-specific manner. For example, function has been attributed to its binding and stabilization of the Myc [19] and Nop2 [17] proteins in breast and hepatocellular carcinoma, respectively. In gastric cancer cells, acts to repress the expression of p15 and p16 via its physical interaction with the polycomb group protein, EZH2 [20]. Also via EZH2 recruitment and regulation of thyroid-stimulating hormone receptor, induces proliferation of thyroid cancer cells [21]. Finally, computational analysis of suggests that it may act via binding and sequestration of mir-200 family members in breast cancer tissue [14]. Due to their complexity and breadth of results, these studies emphasize the importance of disease-specific investigation of mechanisms. Our interest in originated from our.

POMP expression was then modulated using hereditary and pharmacologic methods to determine the consequences about proteasome inhibitor sensitivity in cell lines and choices

POMP expression was then modulated using hereditary and pharmacologic methods to determine the consequences about proteasome inhibitor sensitivity in cell lines and choices. (or using either brief hairpin (sh) RNAs or a pharmacologic agent restored level of sensitivity in cell lines, major plasma cells, and an myeloma model. Experimental Methods Cell Lines and Major Examples Drug-naive and bortezomib-resistant myeloma cell lines had been developed and taken care of as referred to previously (27). Cell range authentication was performed by our cell range characterization primary using brief tandem do it again profiling. Bortezomib was taken off the tradition for at least seven days ahead of all tests, unless indicated in any other case, to negate the chance that proteasome inhibitor-induced oxidative tension was impacting and manifestation. Major plasma cells had been purified from bone tissue marrow aspirates gathered from individuals under an authorized protocol through the Institutional Review Panel at the next Affiliated Medical center of Soochow College or university after educated consent was acquired in compliance using the Declaration of Helsinki. The medical background, including prior remedies, of the individuals whose samples had been utilized is demonstrated in Desk 1. TABLE 1 Features of myeloma individuals whose major plasma cells had Encequidar mesylate been studied The next abbreviations are utilized: M, male; F, feminine; ISS, International Staging Program; DS, Durie-Salmon Staging Program; VAD, vincristine, doxorubicin, and dexamethasone; MP, prednisone plus melphalan; PAD, bortezomib, doxorubicin, plus dexamethasone; VMP, bortezomib, melphalan, and prednisone; VDT, bortezomib, dexamethasone, and thalidomide; Dvd and blu-ray, pegylated liposomal doxorubicin, vincristine, Rabbit Polyclonal to EDG7 and dexamethasone; MPR, melphalan, prednisone, and lenalidomide; M2, carmustine, vincristine, cyclophosphamide, melphalan, and prednisone. a, 2 weeks before test collection; b, 4 weeks before test collection; c, six months before test collection; d, six months before test collection; e, 12 months before test collection; f, 4 years before test collection. (FAMTM), (FAMTM), proteasome subunit, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, VIC?) TaqMan gene manifestation as multiplexed assays, triplicate samples on the StepOnePlus PCR program (Applied Encequidar mesylate Biosystems). Comparative quantification was completed using the comparative technique after normalization to the inner GAPDH control, where almost all samples had been normalized to wild-type or vehicle controls after that. NRF2 and POMP Silencing Six lentivirus-based shRNAs geared to shRNA lentiviral vectors included two target-specific constructs, CCGGCTATTGGATTTGAGGATATTCCTCGAGGAATATCCTCAAATCCAATAGTTTTTG and CCGGGGGTCTATTTGCTCCGCTAAACTCGAGTTTAGCGGAGCAAATAGACCCTTTTTG. shRNA lentiviral vectors included two target-specific constructs, CCGGCCGGCATTTCACTAAACACAACTCGAGTTGTGTTTAGTGAAATGCCGGTTTTT and CCGGGCACCTTATATCTCGAAGTTTCTCGAGAAACTTCGAGATATAAGGTGCTTTTT.Sequences from build 3 were in that case also found in some transient transfection assays to knock straight down without subsequent Encequidar mesylate antibiotic selection. Non-targeting shRNAs Encequidar mesylate (KO-NT) or shRNAs Encequidar mesylate focusing on (KO-3) were released by electroporation using the Neon? transfection program (Thermo Fisher Scientific). POMP and NRF2 Manifestation pCMV6-XL5 vectors including or cDNAs had been bought from OriGene (Rockville, MD). or was subcloned in to the lentiviral vector transfer plasmid pCDH-CMV-MCS-EF1-coGFP to create pCDH-CMV-NRF2-EF1-coGFP or pCDH-CMV-POMP-EF1-coGFP. The recombinant pCDH-CMV-POMP-EF1-coGFP vector, pCDH-CMV-NRF2-EF1-coGFP vector, or the control vector pCDH-CMV-MCS-EF1-coGFP was transfected using the product packaging vectors psPAX2 and pMD2.G into 293T cells by calcium mineral chloride to create lentiviruses. KAS-6/1 and OPM-2 cells had been contaminated with control or either TK control create and clear pCMV6-XL5 vector or pCMV6-XL5-NRF2 by electroporation using the Neon? transfection program (Thermo Fisher Scientific). The luciferase/luciferase percentage was determined to normalize for transfection effectiveness. Electrophoretic Mobility Change Assay DNA-protein binding assays had been completed with nuclear draw out from KAS-6/1 V10R cells with 3-biotinylated artificial complementary oligonucleotides (Sigma). The series from the oligonucleotide utilized was 5-CTCCAGCCTAGGTGACACAGCAAGA-3, as well as the tagged oligonucleotides had been annealed by combining equal molar levels of both single-stranded oligonucleotides, heating system to 95 C for 5 min, accompanied by ramp chilling to 25 C over an interval of 45 min. Nuclear components were ready using the nuclear/cytosol fractionation package (BioVision, Carlsbad, CA) following a manufacturer’s guidelines. Binding reactions had been completed for 20 min at space temperature in the current presence of 50 ng/l poly(dI-dC), 0.05% Nonidet P-40, 5 mm MgCl2, 10 mm EDTA, and 2.5% glycerol in 1 binding buffer using 20 fmol of biotin end-labeled focus on DNA and 4 g.

Further analyses of the mechanisms that regulate CD138 expression and related biological processes including cell adhesion or drug sensitivity should contribute not only to a better understanding of the disease, but also to an improvement of the prognosis of myeloma

Further analyses of the mechanisms that regulate CD138 expression and related biological processes including cell adhesion or drug sensitivity should contribute not only to a better understanding of the disease, but also to an improvement of the prognosis of myeloma. Acknowledgments This study was supported by a grant from your Amyloidosis Research Committee for Research on Intractable Diseases from your fallotein Ministry of Health, Labour and Welfare, Japan.. those of CD20, CXCR4 and B cell-specific transcription factors increased compared with those under normoxic conditions. Stem cell-specific transcription factors were upregulated under hypoxic conditions, while no difference was observed in ALDH activity. The reduced CD138 expression under hypoxic conditions recovered when cells were treated with ATRA, even under hypoxic conditions, along with decreases in the expression of stem cell-specific transcription factor. Interestingly, ATRA treatment sensitized MM cells to bortezomib under hypoxia. We propose that hypoxia induces immature and stem cell-like transcription phenotypes in myeloma cells. Taken together with our previous observation that decreased CD138 expression is usually correlated with disease progression, the present data suggest that a hypoxic microenvironment affects the phenotype of MM cells, which may correlate with disease progression. (3) reported that myeloma stem cells are enriched in the CD138-negative populace. During normal B-cell development, abundant CD138 (also known as syndecan-1: SDC1) expression is highly specific for terminally differentiated plasma cells in the bone marrow (4). Since CD138 expression is also a hallmark of malignant plasma cells (myeloma cells), it has been utilized for myeloma cell purification (5) and is considered to be a target for treatment (6). While the majority of myeloma cells express CD138, decreased expression of CD138 is occasionally found in clinical practice (7C9). Even though association between CD138 expression and myeloma stem cells remains a matter of argument (10), several reports have shown that CD138-low or -unfavorable myeloma cells may contribute to drug resistance or relapse of the disease (9,11,12). Therefore, analysis of CD138 downregulation in myeloma cells is required for a better understanding of myeloma biology. Previous reports Gemfibrozil (Lopid) have indicated that this bone marrow microenvironment may contribute to CD138 downregulation (13C16). Among numerous factors in the tumor microenvironment, hypoxia is one of the important factors associated with tumor progression, poor clinical outcomes, dedifferentiation, and formation of malignancy stem cell niches in solid tumors (17). Based on recent findings showing a correlation of MM at the advanced stage with hypoxic conditions in the microenvironment within the bone marrow (18), we hypothesized that CD138 expression may be influenced by hypoxia. In the present study, we compared the changes in CD138 and various transcription factor expressions in myeloma cells under hypoxic or normoxic conditions. We also attempted to Gemfibrozil (Lopid) revert CD138 expression in cells under hypoxia by treatment with all-trans retinoic acid (ATRA). The influence of ATRA around the sensitivity to bortezomib under hypoxic conditions was also examined. Materials and methods Cell culture Human myeloma cell lines, KMS-12BM (19) and RPMI 8226 (20), were Gemfibrozil (Lopid) obtained from the Health Science Research Resources Lender (Osaka, Japan) and managed in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37C under 5% CO2. The two myeloma cell lines were cultured under normoxic (21% O2) and hypoxic (1% O2) conditions for up to 30 days, with new medium provided every 3 days. Experiments under hypoxic conditions were performed in a Personal CO2 Multigas Incubator (ASTEC, Fukuoka, Japan). Circulation cytometric analysis of surface antigens MM cell lines cultured under normoxic and hypoxic conditions were stained with the following fluorescently-labeled antibodies: FITCCD138 (clone MI15), FITC-CD38 (clone HIT2), PE-CD44 (clone 515), PE-CD45 (clone HI30), FITC-CD49d (clone gf10) (BD Biosciences, Franklin Lakes, NJ, Gemfibrozil (Lopid) USA); PE-CD54 (clone HCD54), PE-CXCR4 (clone 12G5), PE-MDR-1 (clone UIC2), APC-ABCG2 (clone 5D3) (Biolegend, San Diego, CA, USA); FITC-CD19 (clone HD37), FITC-CD20 (clone B-Ly1) (Dako, Glostrup, Denmark); and Alexa 647-CS1 (clone 162) (AbD Serotec, Oxford, UK). Density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden), the forward/side scatter profile and 7-amino-actinomycin D (7-AAD) (BD Biosciences).