搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Here,we present a protocol for isolating and culturing mouse photoreceptors in a minimal,chemically defined medium free from serum. We describe steps for retina dissection,enzymatic dissociation,photoreceptor enrichment,cell culture,extracellular vesicles (EVs) enrichment,and EV ultrastructural analysis. This protocol,which has been verified for cultured cells derived from multiple murine strains,allows for the study of several aspects of photoreceptor biology,including EV isolation and nanotube formation. For complete details on the use and execution of this protocol,please refer to Kalargyrou et al. (2021). 1 Subject areas: Cell Biology,Molecular Biology,Neuroscience View Publication

In dynamic light microscopy applications,imaging specimens from multiple angles while maintaining controlled temperature conditions is crucial for comprehensive and accurate analysis. To address these challenges,we present iCAT,an open-source multifunctional accessory designed to enhance light microscopy. It enables specimen rotation along the axial plane,incorporates built-in modules for precise temperature control,features an integrated LED,and includes a camera for real-time specimen monitoring. It can be easily 3D-printed and assembled using readily available electrical components. Combined with any up-right microscope,this versatile device allows researchers to capture detailed images and videos of organoid cultures and live or fixed specimens,such as C. elegans,zebrafish,drosophila,or mouse embryos. The potential applications of iCAT in investigating dynamic cellular processes and complex developmental phenomena are vast,inspiring researchers to explore its possibilities and push the boundaries of biological research. iCAT,an open-source,3D-printable microscopy accessory,enables axial specimen rotation,temperature control,and live monitoring for high-resolution imaging of organoids and diverse model organisms. View Publication

Lenalidomide is the first karyotype-selective therapeutic approved for the treatment of myelodysplastic syndromes (MDS) owing to high rates of erythroid and cytogenetic response in patients with chromosome 5q deletion [del(5q)]. Although haploinsufficiency for the RPS14 gene and others encoded within the common deleted region (CDR) have been implicated in the pathogenesis of the del(5q) phenotype,the molecular basis of the karyotype specificity of lenalidomide remains unexplained. We focused our analysis on possible haplodeficient enzymatic targets encoded within the CDR that play key roles in cell-cycle regulation. We show that the dual specificity phosphatases,Cdc25C and PP2Acalpha,which are coregulators of the G(2)-M checkpoint,are inhibited by lenalidomide. Gene expression was lower in MDS and acute myeloid leukemia (AML) specimens with del(5q) compared with those with alternate karyotypes. Lenalidomide inhibited phosphatase activity either directly (Cdc25C) or indirectly (PP2A) with corresponding retention of inhibitory phospho-tyrosine residues. Treatment of del(5q) AML cells with lenalidomide induced G(2) arrest and apoptosis,whereas there was no effect in nondel(5q) AML cells. Small interfering RNA (shRNA) suppression of Cdc25C and PP2Acalpha gene expression recapitulated del(5q) susceptibility to lenalidomide with induction of G(2) arrest and apoptosis in both U937 and primary nondel(5q) MDS cells. These data establish a role for allelic haplodeficiency of the lenalidomide inhibitable Cdc25C and PP2Acalpha phosphatases in the selective drug sensitivity of del(5q) MDS. View Publication

HIV-infected slow progressors (SP) represent a heterogeneous group of subjects who spontaneously control HIV infection without treatment for several years while showing moderate signs of disease progression. Under conditions that remain poorly understood,a subgroup of these subjects experience failure of spontaneous immunological and virological control. Here we determined the frequency of SP subjects who showed loss of HIV control within our Canadian Cohort of HIV(+) Slow Progressors and identified the proinflammatory cytokine IL-32 as a robust biomarker for control failure. Plasmatic levels of the proinflammatory isoforms of IL-32 (mainly β and γ) at earlier clinic visits positively correlated with the decline of CD4 T-cell counts,increased viral load,lower CD4/CD8 ratio and levels of inflammatory markers (sCD14 and IL-6) at later clinic visits. We present here a proof-of-concept for the use of IL-32 as a predictive biomarker for disease progression in SP subjects and identify IL-32 as a potential therapeutic target. View Publication

Intestinal tuft cells are one of 4 secretory cell linages in the small intestine and the source of IL-25,a critical initiator of the type 2 immune response to parasite infection. When Raptor,a critical scaffold protein for mammalian target of rapamycin complex 1 (mTORC1),was acutely deleted in intestinal epithelium via Tamoxifen injection in Tritrichomonas muris (Tm) infected mice,tuft cells,IL-25 in epithelium and IL-13 in the mesenchyme were significantly reduced,but Tm burden was not affected. When Tm infected mice were treated with rapamycin,DCLK1 and IL-25 expression in enterocytes and IL-13 expression in mesenchyme were diminished. After massive small bowel resection,tuft cells and Tm were diminished due to the diet used postoperatively. The elimination of Tm and subsequent re-infection of mice with Tm led to type 2 immune response only in WT,but Tm colonization in both WT and Raptor deficient mice. When intestinal organoids were stimulated with IL-4,tuft cells and IL-25 were induced in both WT and Raptor deficient organoids. In summary,our study reveals that enterocyte specific Raptor is required for initiating a type 2 immune response which appears to function through the regulation of mTORC1 activity. View Publication

Fibroblast growth factor (FGF) signaling plays an important role in embryonic stem cells and adult tissue homeostasis,but the function of FGFs in mammary gland stem cells is less well defined. Both FGFR1 and FGFR2 are expressed in basal and luminal mammary epithelial cells (MECs),suggesting that together they might play a role in mammary gland development and stem cell dynamics. Previous studies have demonstrated that the deletion of FGFR2 resulted only in transient developmental defects in branching morphogenesis. Using a conditional deletion strategy,we investigated the consequences of FGFR1 deletion alone and then the simultaneous deletion of both FGFR1 and FGFR2 in the mammary epithelium. FGFR1 deletion using a keratin 14 promoter-driven Cre-recombinase resulted in an early,yet transient delay in development. However,no reduction in functional outgrowth potential was observed following limiting dilution transplantation analysis. In contrast,a significant reduction in outgrowth potential was observed upon the deletion of both FGFR1 and FGFR2 in MECs using adenovirus-Cre. Additionally,using a fluorescent reporter mouse model to monitor Cre-mediated recombination,we observed a competitive disadvantage following transplantation of both FGFR1/R2-null MECs,most prominently in the basal epithelial cells. This correlated with the complete loss of the mammary stem cell repopulating population in the FGFR1/R2-attenuated epithelium. FGFR1/R2-null MECs were partially rescued in chimeric outgrowths containing wild-type MECs,suggesting the potential importance of paracrine mechanisms involved in the maintenance of the basal epithelial stem cells. These studies document the requirement for functional FGFR signaling in mammary stem cells during development. View Publication

The impact of shipping temperatures and preservation media used during transport of either peripheral blood mononuclear cells (PBMCs) or Jurkat cells was assessed,in view of implementing of a proficiency testing scheme on mononuclear cell viability. Samples were analyzed before and after shipment at different temperatures (ambient temperature,dry ice,and liquid nitrogen) and in different preservation media (serum with cryoprotectant,commercial cryopreservation solution,and room temperature transport medium). Sample quality was assessed by viability assays (Trypan Blue dye exclusion,flow cytometry,Cell Analysis System cell counting (CASY)),and by ELISpot functional assay. The liquid nitrogen storage and shipment were found to be the most stable conditions to preserve cell viability and functionality. However,we show that alternative high quality shipment conditions for viable cells are dry ice shipment and commercial cryopreservation solution. These were also cost-efficient shipment conditions,satisfying the requirements of a proficiency testing scheme for viable mononuclear cells. Room temperature transport medium dramatically and adversely affected the integrity of mononuclear cells. View Publication

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the Slc6a8 gene. The impaired creatine uptake in the brain leads to developmental delays with intellectual disability. We hypothesized that deficient creatine uptake in CTD cerebral cells impact methylation balance leading to alterations of genes and proteins expression by epigenetic mechanism. In this study,we determined the status of nucleic acid methylation in both Slc6a8 knockout mouse model and brain organoids derived from CTD patients’ cells. We also investigated the effect of dodecyl creatine ester (DCE),a promising prodrug that increases brain creatine content in the mouse model of CTD. The level of nucleic acid methylation was significantly reduced compared to healthy controls in both in vivo and in vitro CTD models. This hypo-methylation tended to be regulated by DCE treatment in vivo. These results suggest that increased brain creatine after DCE treatment restores normal levels of DNA methylation,unveiling the potential of using DNA methylation as a marker to monitor the drug efficacy. View Publication

The efficacy of immunotherapy is often hindered by the suppression of immune responses via the tumor microenvironment (TME). The presence of cancer cells forces other proximal non-cancerous cells to support tumor growth and persistence. A clear example of this cancerous-to-non-cancerous communication is represented by the accumulation of myeloid-derived suppressor cells (MDSCs) within the TME. Several studies have convergently shown that the overexpression of DNA-methyl-transferase-1 (DNMT1) in these cells results in protection from necroptosis and enhanced accumulation in vivo. Conversely,targeting DNMT1 through hypo-methylating agents has shown promising therapeutic potential by not only reducing the levels of MDSCs but also enhancing cancer immunogenicity and the efficacy of immune checkpoint inhibitors (ICI). Methods: Murine 4T1 (triple-negative breast cancer (TNBC)) and CT26 (colon carcinoma) cell lines were cultured under standard conditions and used to generate tumor models in BALB/c mice. An oncolytic adenovirus expressing a DNMT1-targeting short hairpin RNA (OAd.shDNMT1) was engineered and validated for DNMT1 knockdown and genome-wide methylation reduction. Small extracellular vesicles (sEVs) were isolated from virus-infected cancer cells and characterized for RNA content and uptake by MDSCs. MDSC differentiation and suppressive function were assessed in vitro using flow cytometry and co-culture assays with murine splenocytes. In vivo,tumor-bearing mice received intratumoral OAd.shDNMT1,systemic decitabine,or immune checkpoint inhibitors (anti-Programmed cell Death protein-1),and tumor growth,immune infiltration,and systemic MDSC levels were evaluated. Results: In this study,we report that,by using virally infected TNBC murine cells as a source for shDNMT1-loaded sEVs,OAd.shDNMT1 successfully reduced MDSC levels in vitro and in vivo. Furthermore,the co-administration with ICI resulted in a significant tumor growth reduction in mice bearing poorly immunogenic TNBC 4T1 cells. Also,our treatment promoted antitumor immunity,prolonged survival,and complete tumor eradication in modestly immunogenic colon CT26 cancer cells. Conclusions: This multifaceted strategy,based on OV-mediated immune stimulation and reduction of MDSC levels via sEVs,may improve clinical outcomes and the success of immuno-based regimens for patients facing MDSC-rich and highly aggressive cancer subtypes. View Publication

Adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1) infection,occurs in 2% to 4% of the HTLV-1 carriers with a long latent period,suggesting that additional alterations participate in the development of ATL. To characterize and identify novel markers of ATL,we examined the expression profiles of more than 12 000 genes in 8 cases of acute-type ATL using microarray. One hundred ninety-two genes containing interleukin 2 (IL-2) receptor alpha were up-regulated more than 2-fold compared with CD4(+) and CD4(+)CD45RO(+) T cells,and tumor suppressor in lung cancer 1 (TSLC1),caveolin 1,and prostaglandin D2 synthase showed increased expression of more than 30-fold. TSLC1 is a cell adhesion molecule originally identified as a tumor suppressor in the lung but lacks its expression in normal or activated T cells. We confirmed ectopic expression of the TSLC1 in all acute-type ATL cells and in 7 of 10 ATL- or HTLV-1-infected T-cell lines. Introduction of TSLC1 into a human ATL cell line ED enhanced both self-aggregation and adhesion ability to vascular endothelial cells. These results suggested that the ectopic expression of TSLC1 could provide a novel marker for acute-type ATL and may participate in tissue invasion,a characteristic feature of the malignant ATL cells. View Publication