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T cell facs analysis

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https://www.readbyqxmd.com/read/29458078/isolation-of-intact-rna-from-murine-cd4-t-cells-after-intracellular-cytokine-staining-and-fluorescence-activated-cell-sorting
#1
Shajo Kunnath-Velayudhan, Steven A Porcelli
Intracellular cytokine staining (ICS) is a powerful method for identifying functionally distinct lymphocyte subsets, and for isolating these by fluorescence activated cell sorting (FACS). Although transcriptomic analysis of cells sorted on the basis of ICS has many potential applications, this is rarely performed because of the difficulty in isolating intact RNA from cells processed using standard fixation and permeabilization buffers for ICS. To address this issue, we compared three buffers shown previously to preserve RNA in nonhematopoietic cells subjected to intracellular staining for their effects on RNA isolated from T lymphocytes processed for ICS...
February 17, 2018: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29448043/estrogen-serum-concentration-affects-blood-immune-cell-composition-and-polarization-in-human-females-under-controlled-ovarian-stimulation
#2
Pardes Habib, Daniela Dreymueller, Benjamin Rösing, Hannes Botung, Alexander Slowik, Adib Zendedel, Shahin Habib, Stefanie Hoffmann, Cordian Beyer
Estrogens modulate the immune system and possess anti-inflammatory properties. In line, immune cells express a variety of estrogen receptors (ER) including ER-alpha and -beta. In the present study, we examined the influence of 17beta-estradiol (E2) serum concentrations on blood leukocyte composition and their ex vivo polarization/activation status by FACS analysis in sub-fertile human females under controlled ovarian stimulation (COS). Using a set of cell-type and polarization-specific markers, we demonstrate that increased 17ß-estradiol (E2) serum concentrations yield an overall increase in leukocytes, neutrophils and monocytes but decreased lymphocytes...
February 12, 2018: Journal of Steroid Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/29432774/pstpip1-controls-immune-synapse-stability-in-human-t-cells
#3
W J M Janssen, V Grobarova, J Leleux, C H Jongeneel, M van Gijn, J M van Montfrans, M Boes
BACKGROUND: PSTPIP1 is a cytosolic adaptor protein involved with T-cell activation, differentiation, and migration. Upon cognate T-cell contact, PSTPIP1 is recruited to surface-expressed CD2, where it regulates f-actin remodeling. An immune synapse (IS) is thereby rapidly formed, consisting of TCR clusters surrounded by a ring of adhesion molecules including CD2. OBJECTIVE: From genetic screening of primary immunodeficiency patients, we identified two mutations in PSTPIP1, R228C and T274M which we further characterized in primary patient T-cells...
February 9, 2018: Journal of Allergy and Clinical Immunology
https://www.readbyqxmd.com/read/29396668/targeting-ataxia-telangiectasia-mutated-and-rad3-related-kinase-atr-in-pten-deficient-breast-cancers-for-personalized-therapy
#4
Nouf Al-Subhi, Reem Ali, Tarek Abdel-Fatah, Paul M Moseley, Stephen Y T Chan, Andrew R Green, Ian O Ellis, Emad A Rakha, Srinivasan Madhusudan
PURPOSE: Phosphate and tensin homolog (PTEN), a negative regulator of PI3K signaling, is involved in DNA repair. ATR is a key sensor of DNA damage and replication stress. We evaluated whether ATR signaling has clinical significance and could be targeted by synthetic lethality in PTEN-deficient triple-negative breast cancer (TNBC). METHODS: PTEN, ATR and pCHK1Ser345 protein level was evaluated in 1650 human breast cancers. ATR blockade by VE-821 was investigated in PTEN-proficient- (MDA-MB-231) and PTEN-deficient (BT-549, MDA-MB-468) TNBC cell lines...
February 2, 2018: Breast Cancer Research and Treatment
https://www.readbyqxmd.com/read/29371108/the-importance-of-area-scaling-with-facs-diva-software
#5
Amy L Hazen, Timothy Bushnell, David L Haviland
With the release and use of the Becton Dickenson FACS Diva Software, the use of Area as the default parameter came into play. As such, the use of area as a calculated parameter, methods were needed to be employed to ensure doublet discrimination and proper display on standard FSC/SSC. Improper setting of forward area scaling can alter the display cell populations. This combined with improper area gating strategy can lead to doublet inclusion which in sorting rare events can compromise sort purity. In extreme cases where area scaling with the individual lasers is ignored, differences can exist between Area and Height where compensation will likely not be optimal, particularly if one parameter - usually height is saturated...
January 21, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29314593/in-vivo-immuno-reactivity-analysis-of-the-porous-3d-cs-sio2-and-cs-sio2-ha-hybrids
#6
Mengxia Guo, Yifan Dong, Jiangwei Xiao, Ruicai Gu, Maochao Ding, Tao Huang, Junhua Li, Naru Zhao, Hua Liao
Inorganic/organic hybrid silica-chitosan scaffolds have promising potential for bone defect repair, due to the controllable mechanical properties, degradation behavior and scaffold morphology. However, the precise in vivo immuno-reactivity of silica-chitosan hybrids with various compositions is still poorly defined. In this study, we fabricated the 3D interconnected porous chitosan-silica (CS/SiO2 ) and chitosan-silica-hydroxyapatite (CS/SiO2 /HA) hybrids, through sol-gel process and 3D plotting skill, followed by the naturally or freeze drying separately...
January 3, 2018: Journal of Biomedical Materials Research. Part A
https://www.readbyqxmd.com/read/29311117/expanded-genomic-profiling-of-circulating-tumor-cells-in-metastatic-breast-cancer-patients-to-assess-biomarker-status-and-biology-over-time
#7
Mark Jesus M Magbanua, Hope S Rugo, Denise M Wolfe, Louai Hauranieh, Ritu Roy, Praveen Pendyala, Eduardo Sosa, Janet H Scott, Jin Sun Lee, Brandelyn N Pitcher, Terry M Hyslop, William T Barry, Steven J Isakoff, Maura N Dickler, Laura J Van't Veer, John W Park
Purpose: We profiled circulating tumor cells (CTCs) patients to study the biology of blood-borne metastasis and to monitor biomarker status in metastatic breast cancer (MBC).Methods: CTCs were isolated from 105 MBC patients using EPCAM-based immunomagnetic enrichment and fluorescence-activated cells sorting (IE/FACs), 28 of whom had serial CTC analysis (74 samples, 2-5 time points). CTCs were subjected to microfluidic-based multiplex QPCR array of 64 cancer-related genes (n=151) and genome-wide copy number analysis by array comparative genomic hybridization (n=49)...
January 8, 2018: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
https://www.readbyqxmd.com/read/29282022/chromosomal-instability-induced-by-increased-birc5-survivin-levels-affects-tumorigenicity-of-glioma-cells
#8
Marina Conde, Susanne Michen, Ralf Wiedemuth, Barbara Klink, Evelin Schröck, Gabriele Schackert, Achim Temme
BACKGROUND: Survivin, belonging to the inhibitor of apoptosis (IAP) gene family, is abundantly expressed in tumors. It has been hypothesized that Survivin facilitates carcinogenesis by inhibition of apoptosis resulting in improved survival of tumorigenic progeny. Additionally, Survivin plays an essential role during mitosis. Together with its molecular partners Aurora B, Borealin and inner centromere protein it secures bipolar chromosome segregation. However, whether increased Survivin levels contribute to progression of tumors by inducing chromosomal instability remains unclear...
December 28, 2017: BMC Cancer
https://www.readbyqxmd.com/read/29250828/cd4-cd25hi-foxp3-regulatory-t-cells-in-long-term-cardiac-xenotransplantation
#9
Avneesh K Singh, Joshua L Chan, Caleb N Seavey, Philip C Corcoran, Robert F Hoyt, Billeta G T Lewis, Marvin L Thomas, David L Ayares, Keith A Horvath, Muhammad M Mohiuddin
BACKGROUND: CD4+CD25Hi FoxP3+ T (Treg) cells are a small subset of CD4+ T cells that have been shown to exhibit immunoregulatory function. Although the absolute number of Treg cells in peripheral blood lymphocytes (PBL) is very small, they play an important role in suppressing immune reactivity. Several studies have demonstrated that the number of Treg cells, rather than their intrinsic suppressive capacity, may contribute to determining the long-term fate of transplanted grafts. In this study, we analyzed Treg cells in PBL of long-term baboon recipients who have received genetically modified cardiac xenografts from pig donors...
December 17, 2017: Xenotransplantation
https://www.readbyqxmd.com/read/29203363/mechanism-of-action-of-g-quadruplex-forming-oligonucleotide-homologous-to-the-telomere-overhang-in-melanoma
#10
Gagan Chhabra, Luke Wojdyla, Mark Frakes, Zachary Schrank, Brandon Leviskas, Marko Ivancich, Pooja Vinay, Ramesh Ganapathy, Benjamin E Ramirez, Neelu Puri
T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3'-telomeric overhang of telomeres, elicits potent DNA-damage responses in melanoma cells; however, its mechanism of action is largely unknown. GROs can form G-quadruplexes (G4) which are stabilized by the hydrogen-bonding of guanine residues. In this study, we confirmed the G4-forming capabilities of T-oligo using non-denaturing PAGE, NMR and immunofluorescence. Using an anti-G-quadruplex antibody (BG4) we showed that T-oligo can form G4 in the nuclei of melanoma cells...
December 1, 2017: Journal of Investigative Dermatology
https://www.readbyqxmd.com/read/29162985/cbfa2t2-is-associated-with-a-cancer-stem-cell-state-in-renal-cell-carcinoma
#11
Du-Chu Chen, You-De Liang, Liang Peng, Yi-Ze Wang, Chun-Zhi Ai, Xin-Xing Zhu, Ya-Wei Yan, Yasmeen Saeed, Bin Yu, Jingying Huang, Yuxin Gao, Jiaqi Liu, Yi-Zhou Jiang, Min Liu, Demeng Chen
Background: Renal cell carcinoma (RCC) is the most common kidney cancer, accounting for approximately 80-90% of all primary kidney cancer. Treatment for patients with advanced RCC remains unsatisfactory. Rare cancer stem cells (CSCs) are proposed to be responsible for failure of current treatment. Methods: OncoLnc was used as a tool for interactively exploring survival correlations. Gene manipulation and expression analysis were carried out using siRNA, RT-PCR and Western blotting...
2017: Cancer Cell International
https://www.readbyqxmd.com/read/29150331/anti-amyloidogenic-and-anti-apoptotic-effect-of-%C3%AE-bisabolol-against-a%C3%AE-induced-neurotoxicity-in-pc12-cells
#12
Balakrishnan Shanmuganathan, Venkatesan Suryanarayanan, Sethuraman Sathya, Muralidharan Narenkumar, Sanjeev Kumar Singh, Kandasamy Ruckmani, Kasi Pandima Devi
Alzheimer's disease (AD) is a life-threatening neurodegenerative disorder leading to dementia, with a progressive decline in memory and other thinking skills of elderly populace. Of the multiple etiological factors of AD, the accumulation of senile plaques (SPs) particularly as Aβ oligomers correlates with the relentlessness cognitive impairment in AD patients and play a vital role in AD pathology. Since natural essential oil constituents have successfully served as a source of drugs for AD treatment, the present study aims at the in vitro and in silico investigation of anti-amyloidogenic potential and anti-apoptotic property of the α-bisabolol against Aβ25-35 induced neurotoxicity in PC12 cells...
October 10, 2017: European Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/29132406/sjcrt-a-recombinant-schistosoma-japonicum-calreticulin-induces-maturation-of-dendritic-cells-and-a-th1-polarized-immune-response-in-mice
#13
Lizhen Ma, Dandan Li, Chunxiu Yuan, Xiangqian Zhang, Na Ta, Xiaochao Zhao, Yumei Li, Xingang Feng
BACKGROUND: It is well known that immunization of radiation-attenuated (RA) schistosoma cercariae or schistosomula can induce high levels of protective immunity against schistosoma cercariae reinfection in many animals. Many studies have shown that the Th1 cellular immune response is crucial for the protective effect elicited by RA schistosomula. However, the molecular mechanism of this strong protective immunity remains unclear. METHODS: The expression profiles of Schistosoma japonicum calreticulin (SjCRT) in RA and normal schistosoma-derived cells were investigated by flow cytometry...
November 13, 2017: Parasites & Vectors
https://www.readbyqxmd.com/read/29126435/human-amnion-cells-reverse-acute-and-chronic-pulmonary-damage-in-experimental-neonatal-lung-injury
#14
Dandan Zhu, Jean Tan, Amina S Maleken, Ruth Muljadi, Siow T Chan, Sin N Lau, Kirstin Elgass, Bryan Leaw, Joanne Mockler, Daniel Chambers, Kristen T Leeman, Carla F Kim, Euan M Wallace, Rebecca Lim
BACKGROUND: Despite advances in neonatal care, bronchopulmonary dysplasia (BPD) remains a significant contributor to infant mortality and morbidity. While human amnion epithelial cells (hAECs) have shown promise in small and large animal models of BPD, there is scarce information on long-term benefit and clinically relevant questions surrounding administration strategy remain unanswered. In assessing the therapeutic potential of hAECs, we investigated the impact of cell dosage, administration routes and timing of treatment in a pre-clinical model of BPD...
November 10, 2017: Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/29091262/isolation-and-functional-use-of-human-nkt-cells
#15
Mark A Exley, S Brian Wilson, Steven P Balk
This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term 'iNKT' derives from the fact that a large fraction of murine and some human NK marker+ T cells ('NKT') recognize the MHC class I-like CD1d protein and use an identical 'invariant' TCRα chain, which is generated in humans by precise Vα24 and Jα18 rearrangements with either no N-region diversity or subsequent trimming to identical or nearly identical amino acid sequence (hence, 'iNKT' cells)...
November 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29073251/single-cell-analysis-identifies-cellular-markers-of-the-hiv-permissive-cell
#16
Sylvie Rato, Antonio Rausell, Miguel Muñoz, Amalio Telenti, Angela Ciuffi
Cellular permissiveness to HIV infection is highly heterogeneous across individuals. Heterogeneity is also found across CD4+ T cells from the same individual, where only a fraction of cells gets infected. To explore the basis of permissiveness, we performed single-cell RNA-seq analysis of non-infected CD4+ T cells from high and low permissive individuals. Transcriptional heterogeneity translated in a continuum of cell states, driven by T-cell receptor-mediated cell activation and was strongly linked to permissiveness...
October 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/29024986/stromal-fibroblasts-from-perimenopausal-endometrium-exhibit-a-different-transcriptome-than-those-from-the-premenopausal-endometrium
#17
David W Erikson, Fatima Barragan, Terhi T Piltonen, Joseph C Chen, Shaina Balayan, Juan C Irwin, Linda C Giudice
Human endometrium undergoes extensive regeneration on a cyclic basis in premenopausal women and likely occurs through the contribution of stem/progenitor cells. Menopause results in the permanent cessation of menstrual cycles and is preceded by perimenopause, a period of several years in which endocrine and biological changes occur and is a period of risk for endometrial proliferative disorders. The objectives of this study were to identify endometrial mesenchymal stem cells (eMSC) and endometrial stromal fibroblasts (eSF) in endometrium of perimenopausal women and perform expression profile analysis of perimenopausal eMSC and eSF to gain insight into the biology of stem/progenitor and lineage cell populations during the transition to menopause...
September 1, 2017: Biology of Reproduction
https://www.readbyqxmd.com/read/29020964/il-27-mediates-hla-class-i-up-regulation-which-can-be-inhibited-by-the-il-6-pathway-in-hla-deficient-small-cell-lung-cancer-cells
#18
Grazia Carbotti, Amin Reza Nikpoor, Paola Vacca, Rosaria Gangemi, Chiara Giordano, Francesco Campelli, Silvano Ferrini, Marina Fabbi
BACKGROUND: Recently, immunotherapy with anti-PD-1 antibodies has shown clinical benefit in recurrent Small Cell Lung Cancer (SCLC). Since anti-PD-1 re-activates anti-tumor Cytotoxic T Lymphocyte (CTL) responses, it is crucial to understand the mechanisms regulating HLA class I, and PD-L1 expression in HLA-negative SCLC. Here we addressed the role of IL-27, a cytokine related to both IL-6 and IL-12 families. METHODS: The human SCLC cell lines NCI-N592, -H69, -H146, -H446 and -H82 were treated in vitro with different cytokines (IL-27, IFN-γ, IL-6 or a soluble IL-6R/IL-6 chimera [sIL-6R/IL-6]) at different time points and analyzed for tyrosine-phosphorylated STAT proteins by Western blot, for surface molecule expression by immunofluorescence and FACS analyses or for specific mRNA expression by QRT-PCR...
October 11, 2017: Journal of Experimental & Clinical Cancer Research: CR
https://www.readbyqxmd.com/read/29016555/interleukin-10-regulated-tumour-tolerance-in-non-small-cell-lung-cancer
#19
Julius Malte Vahl, Juliane Friedrich, Susanne Mittler, Sonja Trump, Lisanne Heim, Katerina Kachler, Liubov Balabko, Nicole Fuhrich, Carol-Immanuel Geppert, Denis Iulian Trufa, Nina Sopel, Ralf Rieker, Horia Sirbu, Susetta Finotto
BACKGROUND: Lung cancer is the most life-threatening cancer type worldwide. Treatment options include surgery, radio- and chemotherapy, as well as the use of immunomodulatory antibodies. Interleukin (IL)-10 is an immunosuppressive cytokine involved in tumour immune escape. METHODS: Immunohistochemistry (IHC) on human lung surgery tissue as well as human tumour cell line cultures, FACS analysis, real-time PCR and experimental lung cancer. RESULTS: Here we discovered a positive correlation between IL-10 and IL-10 receptor (IL-10R) expression in the lung with tumour diameter in patients with lung cancer (non-small cell lung cancer), the most life-threatening cancer type worldwide...
October 10, 2017: British Journal of Cancer
https://www.readbyqxmd.com/read/28951258/ibrutinib-suppresses-alloantibody-responses-in-a-mouse-model-of-allosensitization
#20
Irene Kim, Gordon Wu, Ning-Ning Chai, Andrew S Klein, Stanley Jordan
BACKGROUND: Ibrutinib is a Bruton's tyrosine Kinase (BTK) antagonist that inhibits B cell receptor (BCR) signaling. Complete BTK deficiency is associated with absence of B-cells. Ibrutinb is currently approved by FDA for treatment of B-cell malignancies, including Waldenström macroglobulinaemia. We recently carried out studies to determine if ibrutinib could modify alloantibody responses. MATERIALS AND METHODS: A mouse model of allogenic sensitization using a C57BL/6 mouse as the recipient of a skin allograft from an HLA-A2 transgenic mouse was utilized to examine the effects of ibrutinib on alloantibody responses and B cell effector functions...
September 23, 2017: Transplant Immunology
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