Synaptotagmin-13 Is a Neuroendocrine Marker in Brain, Intestine and Pancreas

Synaptotagmin-13 (Syt13) is an atypical member of the vesicle trafficking synaptotagmin protein family. The expression pattern and the biological function of this Ca2+-independent protein are not well resolved. Here, we have generated a novel Syt13-Venus fusion (Syt13-VF) fluorescence reporter allele to track and isolate tissues and cells expressing Syt13 protein. The reporter allele is regulated by endogenous cis-regulatory elements of Syt13 and the fusion protein follows an identical expression pattern of the endogenous Syt13 protein. The homozygous reporter mice are viable and fertile. We identify the expression of the Syt13-VF reporter in different regions of the brain with high expression in tyrosine hydroxylase (TH)-expressing and oxytocin-producing neuroendocrine cells. Moreover, Syt13-VF is highly restricted to all enteroendocrine cells in the adult intestine that can be traced in live imaging. Finally, Syt13-VF protein is expressed in the pancreatic endocrine lineage, allowing their specific isolation by flow sorting. These findings demonstrate high expression levels of Syt13 in the endocrine lineages in three major organs harboring these secretory cells. Collectively, the Syt13-VF reporter mouse line provides a unique and reliable tool to dissect the spatio-temporal expression pattern of Syt13 and enables isolation of Syt13-expressing cells that will aid in deciphering the molecular functions of this protein in the neuroendocrine system.

Synaptotagmin13 Is A Neuroendocrine Marker In Brain, Intestine And Pancreas

Synaptotagmin13 (Syt13) is an atypical member of the vesicle trafficking Synaptotagmin protein family. The expression pattern and the biological function of this Ca2+-independent protein are not well resolved. Here, we have generated a novel Syt13-Venus fusion (Syt13-VF) fluorescence reporter allele to track and isolate tissues and cells expressing Syt13 protein. The reporter allele is regulated by endogenous cis-regulatory elements of Syt13 and the fusion protein follows an identical expression pattern of the endogenous Syt13 protein. The homozygous reporter mice are viable and fertile. We identify the expression of the Syt13-VF reporter in different regions of the brain with high expression in tyrosine hydroxylase (TH)-expressing and oxytocin-producing neuroendocrine cells. Moreover, Syt13-VF is highly restricted to all enteroendocrine cells in the adult intestine that can be traced in live imaging. Finally, Syt13-VF protein is expressed in the pancreatic endocrine lineage allowing their specific isolation by flow sorting. These findings demonstrate high expression levels of Syt13 in the endocrine lineages in three major organs harboring these secretory cells. Collectively, the Syt13-VF reporter mouse line provides a unique and reliable tool to dissect the spatio-temporal expression pattern of Syt13 and enables isolation of Syt13-expressing cells that will aid in deciphering the molecular functions of this protein in the neuroendocrine system.

Efficient transcriptome profiling across the malaria parasite erythrocytic cycle by flow sorting

Plasmodium falciparum is the most virulent and widespread of the human malaria parasite species. This parasite has a complex life cycle that involves sexual replication in a mosquito vector and asexual replication in a human host. During the 48-hour intraerythrocytic developmental cycle (IDC), parasites develop and multiply through the morphologically distinct ring, trophozoite and schizont stages. Stage-specific transcriptomic approaches have shown gene expression profiles continually change throughout the IDC. Cultures of tightly synchronized parasites are required to capture the transcriptome specific to a developmental stage. However, the most commonly used synchronization methods require lysis of late stages, potentially perturbing transcription, and often do not result in tightly synchronized cultures. To produce complete transcriptome profiles of the IDC a synchronous culture requires frequent sampling over a 48-hour period, this is both time consuming and labor intensive. Here we develop a method to sample the IDC densely by isolating parasites from an asynchronous culture with fluorescence activated cell sorting (FACS). We sort parasites in tight windows of IDC progression based on their DNA/RNA abundance. We confirmed the tight synchronization and stage specificity by light microscopy and RNAseq profiling. We optimized our protocol for low numbers of sorted cells allowing us to rapidly capture transcriptome profiles across the entire IDC from a single culture flask. This methodology will allow malaria stage-specific studies to perform experiments directly from asynchronous cultures with high accuracy and without the need for labor-intensive time-course experiments.

Method for Passive Droplet Sorting after Photo-Tagging

We present a method to photo-tag individual microfluidic droplets for latter selection by passive sorting. The use of a specific surfactant leads to the interfacial tension to be very sensitive to droplet pH. The photoexcitation of droplets containing a photoacid, pyranine, leads to a decrease in droplet pH. The concurrent increase in droplet interfacial tension enables the passive selection of irradiated droplets. The technique is used to select individual droplets within a droplet array as illuminated droplets remain in the wells while other droplets are eluted by the flow of the external oil. This method was used to select droplets in an array containing cells at a specific stage of apoptosis. The technique is also adaptable to continuous-flow sorting. By passing confined droplets over a microfabricated trench positioned diagonally in relation to the direction of flow, photo-tagged droplets were directed toward a different chip exit based on their lateral movement. The technique can be performed on a conventional fluorescence microscope and uncouples the observation and selection of droplets, thus enabling the selection on a large variety of signals, or based on qualitative user-defined features.

Isolation of murine large intestinal crypt cell populations with flow sorting

Here we present a high-resolution sorting protocol for colon crypt stem cells, their daughter cells and mature, differentiated cell types. We used freshly dissected mouse colons and validated intestinal cell surface markers amenable to Flow Activated Cell Sorting (FACS). This 5-7 hour protocol enables isolation of six distinct cryptal cell populations (Stem, AbsPro, SecPDG, Tuft, Ent, and EEC) from any mouse strain/background (Figure 1 and 2) . Downstream analysis of sorted cells (Transcriptomics = bulk RNA-seq and Proteomics = small cell number LC/MS) validated the identity of cell populations. An important strength of this protocol is the independence from any trans-genic labeling of cell types and the flexibility for users to add additional markers for a variety of downstream applications. The absence of proteases during dissociation increases antigen expression resolving the six cell types but also decreases cell yield (Figure 3). The main steps of this protocol include: Tissue Dissection, Tissue Dissociation, Preparation for FACS, and Performing FACS.

P234 Characterisation of human synovial tissue dendritic cells in rheumatoid arthritis patients

Background Dendritic cells (DCs) direct immune responses against pathogens while maintaining tolerance to self-antigens. However, their aberrant activation can lead to autoimmunity and inflammation. DCs consist of two subtypes: plasmacytoid and myeloid DCs. Recently, single-cell sequencing has revealed complex heterogeneity within myeloid DCs. These cells can be divided into CD141pos (DC1), DC2 defined as CD1highCD163neg and DC3 defined as CD1clowCD163high. In addition, a population of CD1cnegCD141negCD16pos (DC4), which share some gene expression with CD16pos monocytes, has been identified. To date, most studies of RA investigated DC precursors in circulation or synovial fluid (SF). However, DCs perform their functions within lymphoid or peripheral tissue. Therefore, in this study, we sought to characterise myeloid DC subsets in synovial tissue (ST) with the prospect of better understanding their role in driving autoimmunity in RA. Methods We developed a flow sorting strategy to characterise the phenotype of distinct myeloid DC subsets in multiple biological compartments (PB, SF, and ST). Ultrasound-guided ST biopsies (RA n = 9; Psoriatic arthritis n = 3 with active disease) were digested with liberase prior to analysis. PB DCs (RA n = 19, Psoriatic arthritis n = 16, healthy donors n = 12), and SF DC (n = 3) were used as comparators. ST, SF and PB DCs were sorted then microRNA, pro-inflammatory and regulatory cytokine expression analysed by amplified qPCR. To evaluate the role of miR-155 in RA CD1cpos DC activation, CD1cpos cells were computationally sub-setted from a scRNAseq synovial dataset comparing synovial tissues of healthy individuals (n = 4) with patients with active RA (n = 7) and those in sustained clinical and ultrasound remission (n = 7). To elucidate the role of miR-155, CD1cpos DC from RA patients (n = 10) were sorted then transfected with miR-155 mimic or control mimic and the cytokines expression and produced were evaluated using quantitative RT-PCR and Luminex, respectively. Results Our data revealed that whilst all myeloid DCs subsets can be present in inflamed RA synovium they occur with variable frequencies. CD1cpos (DC2/3) being the most abundant, followed by CD16pos DCs (DC4), whilst CD141pos DCs (DC1) occur in low numbers or are absent. CD1cpos DC sorted from RA ST express high levels of epigenetic regulators of inflammatory response miR-155, IL-6, TNF-a and IL-23 as compared to circulating cells.Unsupervised clustering of synovial CD1c cells identified 4 separate clusters including a distinctive miR155 positive CD1c cluster in RA patients. These cells showed significantly higher expression of pro-inflammatory cytokines and co-stimulatory molecules compared to other synovial CD1c clusters. Overexpression of miR-155 in CD1cpos leads to increased production of TNF-a and IL-23. Conclusion Our data show that miR-155 is strongly implicated as an epigenetic regulator of the pro-inflammatory synovial CD1cpos DCs sub-cluster and contributes to exacerbating RA. Disclosures A. Elmesmari None. L. MacDonald None. D. Vaughan None. B. Tolusso None. L. Bui None. M. Gigante None. F. Federico None. G. Ferraccioli None. E. Gremese None. I. B McInnes None. S. Alivernini None. M. Kurowska-Stolarska None.

Detection of circulating tumour cells in the breast cancer using CytoTrack system

SummaryIntroduction: Plants are a rich source of healing substances. Cancer is a leading cause of death worldwide while breast cancer is the most common cancer among women. Circulating tumour cells (CTCs) are potential founder cells for metastasis. Therefore, their assessment may be used for monitoring of treatment as well as detecting cancer metastatis. Hence, it is suggested that the number of CTCs may be a valuable tumour biomarker during therapy.Objective: The purpose of this study was to detect CTCs in breast cancer and to validate the method of assessment of CTC count using CytoTrack CT11 technology.Methods: MCF-7 cells were sorted by a FACSARIA flow cytometer from blood samples derived from patients who have not been diagnosed with cancer. Identification and quantitative assessment of MCF-7 cells in blood samples were determined by flow sorting. Then, blood samples containing MCF-7 cells or without MCF-7 were scanned with the use of an automated fluorescence scanning microscope.Results: In in vitro model analysing the glass CytoDisc™ with stained MCF-7 cells, we noted the correlation between the amount of observed tumour cells and expected number of tumour cells. Moreover, coefficient of variation in case of the recovery rate of the assumed number of MCF-7 cells was 30%, 17%, 18% and 15%, respectively.Conclusion: Our study suggest that CTCs could be predictive factor in patients with metastatic cancer especially in breast cancer.