Flow Cytometry Cell Sorting Research Articles

Functional BKCa channel in human resident cardiac stem cells expressing W8B2.

Recently, a new population of resident cardiac stem cells (CSCs) positive for the W8B2 marker has been identified. These CSCs are considered to be an ideal cellular source to repair myocardial damage after infarction. However, the electrophysiological profile of these cells has not been characterized yet. We first establish the conditions of isolation and expansion of W8B2+ CSCs from human heart biopsies using a magnetic sorting system followed by flow cytometry cell sorting. These cells display a spindle-shaped morphology, are highly proliferative, and possess self-renewal capacity demonstrated by their ability to form colonies. Besides, W8B2+ CSCs are positive for mesenchymal markers but negative for hematopoietic and endothelial ones. RT-qPCR and immunostaining experiments show that W8B2+ CSCs express some early cardiac-specific transcription factors but lack the expression of cardiac-specific structural genes. Using patch clamp in the whole-cell configuration, we show for the first time the electrophysiological signature of BKCa current in these cells. Accordingly, RT-PCR and western blotting analysis confirmed the presence of BKCa at both mRNA and protein levels in W8B2+ CSCs. Interestingly, BKCa channel inhibition by paxilline decreased cell proliferation in a concentration-dependent manner and halted cell cycle progression at the G0/G1 phase. The inhibition of BKCa also decreased the self-renewal capacity but did not affect migration of W8B2+ CSCs. Taken together, our results are consistent with an important role of BKCa channels in cell cycle progression and self-renewal in human cardiac stem cells.

Flow Cytometry (FCM) Analysis and Fluorescence-Activated Cell Sorting (FACS) of Erythroid Cells.

To study the process of erythropoiesis, it is important to be able to isolate erythroid progenitors and erythroblasts at distinct stages of development. During the past decade, considerable progress has been made on the development of flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) methods for the analysis and isolation of both murine and human erythroid cells at distinct stages of erythropoiesis, based on changes in the expression of cell surface markers. A method for the identification of murine BFU-E and CFU-E cells was reported by Flygare et al., by negative selection for Ter119, B220, Mac-1, CD3, Gr1, Sca-1, CD16/CD32, CD41, and CD34 cells, followed by separation based on the expression levels of CD71. We developed an alternative method in which Ter119 is used as an erythroid lineage marker, and in conjunction with CD44 and cell size as differentiation markers, it is possible to unambiguously distinguish erythroblasts at each developmental stage during murine terminal erythroid differentiation. We also developed methods for the analysis and isolation of human erythroid cells at all developmental stages. BFU-E and CFU-E are characterized by CD45+GPA-IL-3R-CD34+CD36-CD71low and CD45+GPA-IL-3R-CD34-CD36+CD71high phenotypes, respectively; the combination of GPA, band 3 and α4-integrin are used to isolate erythroid cells at all of the terminal stages of human erythropoiesis, including proerythroblasts, early basophilic, late basophilic, polychromatic and orthochromatic erythroblasts.

Fattigation-platform nanoparticles using apo-transferrin stearic acid as a core for receptor-oriented cancer targeting

A major hurdle in cancer treatment is the precise targeting of drugs to the cancer site. As many cancer cells overexpress the transferrin receptor (TfR), the transferrin (Tf)-TfR interaction is widely exploited to target cancer cells. In this study, novel amphiphilic apo-Tf stearic acid (TfS) conjugates were prepared and characterized by Fourier transform infrared (FTIR) spectroscopy, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, and trinitrobenzenesulfonic acid (TNBS) assay. The prepared TfS conjugates were readily self-assembled in water to form nanoparticles (NPs), consisting of TfS as a core of NPs, whose sizes and zeta potentials were determined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and a particle size analyzer. Hydrophilic water-soluble doxorubicin (DOX) was chosen as a model drug. DOX-loaded TfS NPs (NP+DOX), prepared by the adsorption of DOX on the NP surface via the incubation method, were analyzed for their cell targeting and killing efficiencies in TfR-overexpressing A549 and HCT116 cell lines by MTT assay, confocal microscopy, and fluorescence assisted cell sorting (flow cytometry). The data showed that NP+DOX exhibited improved cancer cell targeting and killing properties compared to that reported for free DOX. Further, the cytotoxic efficiency of NP+DOX was comparable to that of PEGylated liposomal product, Doxil®, while its cellular uptake was higher than that of Doxil®. Thus, this novel receptor-based TfS NP drug delivery system has great potential to target TfR-overexpressing cancer cells without off-target effects.

Characterization of human fetal brain endothelial cells reveals barrier properties suitable for in vitro modeling of the BBB with syngenic co-cultures

Endothelial cells (ECs) form the basis of the blood–brain barrier (BBB), a physical barrier that selectively restricts transport into the brain. In vitro models can provide significant insight into BBB physiology, mechanisms of human disease pathology, toxicology, and drug delivery. Given the limited availability of primary human adult brain microvascular ECs (aBMVECs), human fetal tissue offers a plausible alternative source for multiple donors and the opportunity to build syngenic tri-cultures from the same host. Previous efforts to culture fetal brain microvascular ECs (fBMVECs) have not been successful in establishing mature barrier properties. Using optimal gestational age for isolation and flow cytometry cell sorting, we show for the first time that fBMVECs demonstrate mature barrier properties. fBMVECs exhibited similar functional phenotypes when compared to aBMVECs for barrier integrity, endothelial activation, and gene/protein expression of tight junction proteins and transporters. Importantly, we show that tissue used to culture fBMVECs can also be used to generate a syngenic co-culture, creating a microfluidic BBB on a chip. The findings presented provide a means to overcome previous challenges that limited successful barrier formation by fBMVECs. Furthermore, the source is advantageous for autologous reconstitution of the neurovascular unit for next generation in vitro BBB modeling.

KDM5A promotes proliferation and EMT in ovarian cancer and closely correlates with PTX resistance.

The authors initially performed reverse transcription‑quantitative polymerase chain reaction to determine the expression profile of KDM5A in ovarian cancer tissues and adjacent normal tissue. Compared with adjacent normal tissue, it was identified that KDM5A was highly expressed in ovarian cancer tissues. Moreover, human ovarian cell lines also confirmed that KDM5A was highly expressed in ovarian cancer. KDM5A was especially highly expressed in SKOV3/paclitaxel (PTX) cells, which are resistant to PTX. Previous studies demonstrated that chemoresistance in cancer cells facilitates epithelial‑to‑mesenchymal transition (EMT). Following this, whether KDM5A influenced EMT and metastasis was investigated. The expression of KDM5A and N‑cadherin were obviously higher in SKOV3/PTX cells than in SKOV3 cells. The expression of E‑cadherin was decreased and the expression of N‑cadherin was increased following ectopic expression of KDM5A, while the expression of E‑cadherin was increased and the expression of N‑cadherin was decreased following KDM5A depletion. Transwell and wound healing assays were used to explore the function of KMD5A in metastasis. The present results indicated that KDM5A facilitated EMT and metastasis in ovarian cells. Moreover, it was identified that P‑glycoprotein was increased while KDM5A was expressed ectopically in SKOV3 cells. Following fluorescence‑activated cell sorting flow cytometry analysis and CCK‑8 assay all revealed that KDM5A regulated the PTX sensitivity in SKOV3 and SKOV3/PTX cells. In brief, KDM5A is a crucial oncogene that is significantly upregulated in ovarian cancer. Its expression is closely correlated with cancer cell proliferation, EMT and metastasis. KDM5A suppresses ovarian cancer cell apoptosis under PTX treatment.

Phenotypic plasticity in freshwater picocyanobacteria.

Picocyanobacteria can occur as single-cell (Pcy) or as colonies (CPcy). Published evidence suggests that some Pcy strains have the capability to aggregate under certain culture conditions, however this has not been demonstrated to occur in natural environments. We investigated whether the Pcy and CPcy belong to the same species (i.e. phylotype), and the factors that determine their morphological and genetic variability in a hypertrophic shallow lake dominated by picocyanobacteria. Six main different morphologies and >30 phylotypes were observed. All sequences retrieved belonged to the 'Anathece + Cyanobium' clade (Synechococcales) that are known to have the capability of aggregation/disaggregation. The temporal variation of picocyanobacteria morphotype composition was weakly correlated with the DGGE temporal pattern, and could be explained by the composition of the zooplankton assemblage. Laboratory experiments confirmed that the small cladoceran Bosmina favoured the dominance of CPcy, i.e. Cyanodictyon doubled the size of the colonies when present, most likely through the aggregation of single-cell picocyanobacteria into colonies. Flow cytometry cell sorting and 16S rRNA + ITS sequencing of the Pcy and CPcy cytometrically-defined populations revealed that some phylotypes could be found in both sorted populations, suggesting phenotypic plasticity in which various Synechococcales phylotypes could be found in situ either as single-cells or as colonies.

Functional BKCa Channel in Human Resident Cardiac Stem Cells Expressing W8B2

The adult human heart harbors several populations of cardiac/progenitors stem cells that express specific markers. Recently, a new population of resident cardiac stem cells was identified. These cells are positive for W8B2 marker (called also mesenchymal stem cell antigen-1). W8B2 positive cardiac stem cells (W8B2+ CSCs) exhibit a strong therapeutic potential when transplanted into a chronic myocardial infarction rat model. However, the functional characterization (electrophysiology and calcium signaling) of these cells has not been studied yet. We first establish the conditions of isolation and expansion of W8B2+ CSCs from human heart biopsies by magnetic cell sorting system followed by flow cytometry cell sorting. These cells have a self-renewal capacity demonstrated by their ability to form colonies. In addition, our preliminary results of RT-qPCR show an induction of transcripts encoding contractile proteins such as actin and cardiac troponin-T after in vitro differentiation of W8B2+ CSCs. This differentiation is accompanied by the appearance of cyclic calcium activity assessed by the calcium protein sensor GCaMP protein. The analysis of calcium activity shows that calcium oscillations profile change during differentiation. Using patch-clamp in whole cell configuration, we show for the first time the electrophysiological signing of BKCa channel. In addition, RT-PCR analysis reveals the presence of KCNMA1 (BKCa) mRNA in W8B2+ CSCs. Interestingly, BKCa channel inhibition by paxilline decreases cell proliferation in a concentration-dependent manner and abolishes calcium activity after W8B2+ CSCs differentiation. Taken together, our results are consistent with an important role of BKCa channels on cell cycle progression and on calcium activity during stem cell differentiation.

Characterization and Purification of Neoplastic Cells of Nodular Lymphocyte Predominant Hodgkin Lymphoma from Lymph Nodes by Flow Cytometry and Flow Cytometric Cell Sorting

We report the flow cytometric (FC) identification and characterization of lymphocyte predominant (LP) cells from tissues involved by nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). First, we immunophenotyped the NLPHL cell line (DEV) confirming a germinal center immunophenotype, lack of expression of CD32 and CD58, and expression of CD54. Nineteen of 26 lymph nodes involved by NLPHL demonstrated a population with an LP immunophenotype (73%), which included expression of germinal center markers (CD75/Bcl-6-positive, CD32-weak/negative without CD10), a B-cell immunophenotype (CD19/CD20/CD40+), IgD and/or IgM expression (67%), and lack of programmed death-ligand 1/ligand 2. The LP cells demonstrated an adhesion macromolecule expression pattern distinct from Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma (CHL) (uniform CD50 and variable CD58 for NLPHL; minimal CD50, bright CD58 expression for CHL). A two-tube consensus assay identified LP cells in all seven NLPHL cases examined and only one non-NLPHL case (94 cases evaluated). Finally, FC cell sorting studies confirm that FC-defined populations have an LP cytomorphology. Taken together, these findings demonstrate a two-tube consensus assay can be used to immunophenotype NLHPL with high specificity and sensitivity and rapidly purify LP cells for genetic studies. This study also confirms aneuploidy in LP cells, provides antigens that may be helpful in distinguishing NLPHL from CHL, and suggests that Tcells interact less avidly with LP cells than with Hodgkin and Reed-Sternberg cells.

Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening.

When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2 production in biological systems. Here, we re-engineered the natural H2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H2 production levels was found for all automatically selected strains. One of the selected H2-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.

A method for isolation of cone photoreceptors from adult zebrafish retinae

BackgroundCone photoreceptors are specialised sensory retinal neurons responsible for photopic vision, colour perception and visual acuity. Retinal degenerative diseases are a heterogeneous group of eye diseases in which the most severe vision loss typically arises from cone photoreceptor dysfunction or degeneration. Establishing a method to purify cone photoreceptors from retinal tissue can accelerate the identification of key molecular determinants that underlie cone photoreceptor development, survival and function. The work herein describes a new method to purify enhanced green fluorescent protein (EGFP)-labelled cone photoreceptors from adult retina of Tg(3.2gnat2:EGFP) zebrafish.ResultsMethods for dissecting adult zebrafish retinae, cell dissociation, cell sorting, RNA isolation and RNA quality control were optimised. The dissociation protocol, carried out with ~30 retinae from adult zebrafish, yielded approximately 6 × 106 cells. Flow cytometry cell sorting subsequently distinguished 1 × 106 EGFP+ cells and 4 × 106 EGFP− cells. Electropherograms confirmed downstream isolation of high-quality RNA with RNA integrity number (RIN) >7.6 and RNA concentration >5.7 ng/µl obtained from both populations. Reverse Transcriptase-PCR confirmed that the EGFP-positive cell populations express known genetic markers of cone photoreceptors that were not expressed in the EGFP-negative cell population whereas a rod opsin amplicon was only detected in the EGFP-negative retinal cell population.ConclusionsThis work describes a valuable adult zebrafish cone photoreceptor isolation methodology enabling future identification of cone photoreceptor-enriched genes, proteins and signalling networks responsible for their development, survival and function. In addition, this advancement facilitates the identification of novel candidate genes for inherited human blindness.

Molecular markers of putative spermatogonial stem cells in the domestic cat

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

Transfer of diazotroph-derived nitrogen towards non-diazotrophic planktonic communities: a comparative study between <i>Trichodesmium</i> <i>erythraeum</i>, <i>Crocosphaera watsonii</i> and <i>Cyanothece</i> sp.

Abstract. Biological dinitrogen (N2) fixation is the major source of new nitrogen (N) for the open ocean, and thus promotes marine productivity, in particular in the vast N-depleted regions of the surface ocean. Yet, the fate of the diazotroph-derived N (DDN) in marine ecosystems is poorly understood, and its transfer to auto- and heterotrophic surrounding plankton communities is rarely measured due to technical limitations. Moreover, the different diazotrophs involved in N2 fixation (Trichodesmium spp. vs. UCYN) exhibit distinct patterns of N2 fixation and inhabit different ecological niches, thus having potentially different fates in the marine food webs that remain to be explored. Here we used nanometer scale secondary ion mass spectrometry (nanoSIMS) coupled with 15N2 isotopic labelling and flow cytometry cell sorting to examine the DDN transfer to specific groups of natural phytoplankton and bacteria during artificially induced diazotroph blooms in New Caledonia (southwestern Pacific). The fate of the DDN was compared according to the three diazotrophs: the filamentous and colony-forming Trichodesmium erythraeum (IMS101), and the unicellular strains Crocosphaera watsonii WH8501 and Cyanothece ATCC51142. After 48 h, 7–17 % of the N2 fixed during the experiment was transferred to the dissolved pool and 6–12 % was transferred to non-diazotrophic plankton. The transfer was twice as high in the T. erythraeum bloom than in the C. watsonii and Cyanothece blooms, which shows that filamentous diazotrophs blooms are more efficient at promoting non-diazotrophic production in N-depleted areas. The amount of DDN released in the dissolved pool did not appear to be a good indicator of the DDN transfer efficiency towards the non-diazotrophic plankton. In contrast, the 15N-enrichment of the extracellular ammonium (NH4+) pool was a good indicator of the DDN transfer efficiency: it was significantly higher in the T. erythraeum than in unicellular diazotroph blooms, leading to a DDN transfer twice as efficient. This suggests that NH4+ was the main pathway of the DDN transfer from diazotrophs to non-diazotrophs. The three simulated diazotroph blooms led to significant increases in non-diazotrophic plankton biomass. This increase in biomass was first associated with heterotrophic bacteria followed by phytoplankton, indicating that heterotrophs took the most advantage of the DDN in this oligotrophic ecosystem.

Diazotroph derived nitrogen supports diatom growth in the South West Pacific: A quantitative study using nanoSIMS

Nitrogen is essential for life but is often a major limiting nutrient for growth in the ocean. Biological dinitrogen fixation is a major source of new nitrogen to surface waters and promotes marine productivity. Yet the fate of diazotroph-derived nitrogen (DDN) in marine ecosystems has been poorly studied, and its transfer to auto- and heterotrophic plankton has not been measured. Here, we use high-resolution nanometer scale secondary ion mass spectrometry (nanoSIMS) coupled with N-15(2) isotopic labelling and flow cytometry cell sorting to examine the DDN transfer to specific groups of natural phytoplankton and bacteria during three diazotroph blooms dominated by the cyanobacterium Trichodesmium spp. in the South West Pacific. During these experiments, 13%+/- 2% to 48%+/- 5% of the fixed N-15(2) was released into the dissolved pool and 6%+/- 1% to 8%+/- 2% of this DDN was transferred to non-diazotrophic plankton after 48 h. The primary beneficiaries of this DDN were diatoms (45%+/- 4% to 61%+/- 38%) and bacteria (22%+/- 27% to 38%+/- 12%), followed by pico-phytoplankton (3%+/- 1% to 21%+/- 14%). The DDN was quickly converted to non-diazotrophic plankton biomass, in particular that of diatoms, which increased in abundance by a factor of 1.4-15 over the course of the three experiments. The single-cell approach we used enabled quantification of the actual transfer of DDN to specific groups of autotrophic and heterotrophic plankton in the surface ocean, revealing a previously unseen level of complexity in the pathways that occur between N-2 fixation and the eventual export of DDN from the photic zone.

18F-FCWAY, a serotonin 1A receptor radioligand, is a substrate for efflux transport at the human blood-brain barrier

Efflux transporters at the blood–brain barrier can decrease the entry of drugs and increase the removal of those molecules able to bypass the transporter. We previously hypothesized that 18F-FCWAY, a radioligand for the serotonin 5-HT1A receptor, is a weak substrate for permeability glycoprotein (P-gp) based on its very early peak and rapid washout from human brain. To determine whether 18F-FCWAY is a substrate for P-gp, breast cancer resistance protein (BCRP), and multidrug resistance protein (MRP1) — the three most prevalent efflux transporters at the blood–brain barrier — we performed three sets of experiments. In vitro, we conducted fluorescence-activated cell sorting (FACS) flow cytometry studies in cells over-expressing P-gp, BCRP, and MRP1 treated with inhibitors specific to each transporter and with FCWAY. Ex vivo, we measured 18F-FCWAY concentration in plasma and brain homogenate of transporter knockout mice using γ-counter and radio-HPLC. In vivo, we conducted positron emission tomography (PET) studies to assess changes in humans who received 18F-FCWAY during an infusion of tariquidar (2–4mg/kg iv), a potent and selective P-gp inhibitor. In vitro studies showed that FCWAY allowed fluorescent substrates to get into the cell by competitive inhibition of all three transporters at the cell membrane. Ex vivo measurements in knockout mice indicate that 18F-FCWAY is a substrate only for P-gp and not BCRP. In vivo, tariquidar increased 18F-FCWAY brain uptake in seven of eight subjects by 60–100% compared to each person's baseline. Tariquidar did not increase brain uptake via some peripheral mechanism, given that it did not significantly alter concentrations in plasma of the parent radioligand 18F-FCWAY or its brain-penetrant radiometabolite 18F-FC. These results show that 18F-FCWAY is a weak substrate for efflux transport at the blood–brain barrier; some radioligand can enter brain, but its removal is hastened by P-gp. Although 18F-FCWAY is not ideal for measuring 5-HT1A receptors, it demonstrates that weak substrate radioligands can be useful for measuring both increased and decreased function of efflux transporters, which is not possible with currently available radioligands such as 11C-loperamide and 11C-verapamil that are avid substrates for transporters.

Specific Single-Cell Isolation of Escherichia coli O157 from Environmental Water Samples by Using Flow Cytometry and Fluorescence-Activated Cell Sorting.

Contamination of food and water with pathogenic bacteria is of concern. Although culture-independent detection and quantification of pathogens is useful, isolation of pathogenic bacteria is still important when identifying the sources of pathogens. Here, we report the use of flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) to specifically detect and isolate individual Escherichia coli O157:H7 cells from water samples. When present at >10 cells/mL water, target pathogen was specifically detected and isolated. The FACS-sorted E. coli O157:H7 population reflected the original population diversity, in contrast to the populations obtained by immunomagnetic separation. Relative abundance of multiple pathogenic strains is important when performing source-tracking studies; therefore, single-cell isolation with FCM-FACS can be a useful tool to obtain pathogenic bacteria for source tracking purpose.

237 - HIF-1α plays a key role in the response of HNSCC cancer stem cells to photon and carbon ion exposures

Purpose: Head and neck squamous cells carcinomas (HNSCC) have a poor prognosis due to escapement to anti-cancer therapies which leads to locoregional recurrences. The presence of cancer stem cells (CSCs), resistant to chemo- and radio-therapies, could explain these resistances and recurrences. Hadrontherapy has demonstrated favorable results for many cancers, since carbon ions induced more CSC’s cell death than photons. However, for the HNSCC tumor, local control remains low. CSCs are located in a hypoxic microenvironment and hypoxia is well-known for being able to induce the epithelial mesenchymal transition (EMT). The aim of this study is to investigate the response of CSCs to radiotherapy, compared with carbon ions under hypoxic conditions. Many signaling pathways involved in the resistance of CSCs and the invasion/migration process depend on the stabilization of HIF-1α. Reactive Oxygen Species (ROS), which are produced under hypoxia and in response to irradiation, play a key role in this stabilization. However, since few data are available, the mechanisms involving ROS production, HIF-1α stabilization as well as the invasion/migration process that follows, need to be clarified in HNSCC CSCs exposed to carbon or photon radiations in a hypoxic environment. Material and methods: Two HNSCC cells lines, SQ20B and FaDu, and their CSCs were grown in normoxic and hypoxic (1% O2) conditions. CSCs were isolated by flow cytometry cell sorting. Cell survival curves were performed in response to photon (250kV) and carbon ion (75MeV/n, GANIL, France) irradiations in order to define the Oxygen Enhancement Ratio (OER). The expression of HIF-1α was followed by Western-Blots. ROS were quantified with a CM-H2DCFDA dye and Migration/Invasion with Boyden chambers. Results: After photons, HNSCC cells and their CSCs appeared more resistant under hypoxia (OER>1.2) whereas the oxygen effects were cancelled after carbon ions (OER=1). Interestingly, the OER values and the expression of HIF-1α seemed to be linked. HIF-1α was weakly expressed after photon irradiation and fully inhibited after carbon ions. Additionally, under hypoxia, HIF-1α expression appeared earlier in CSCs than in the parental cell lines, confirming their adaptive properties to hypoxia. In CSCs, this expression was correlated with ROS levels. As a consequence, since HIF- 1α is known to promote EMT, under normoxia as well as hypoxia, the invasion/migration abilities of CSCs were more important than in non-CSCs. Finally, after carbon ions, either in normoxic or hypoxic conditions, invasion/migration processes were decreased compared with photons. Conclusion: HIF-1α plays a key role in the radioresistance of CSCs and in their invasiveness abilities. No expression of HIF-1α was found after carbon ion exposure suggesting that carbon ions could be a relevant therapeutic alternative to kill CSCs in their microenvironment.

A novel procedure of quantitation of virus based on microflow cytometry analysis.

The accurate and fast titration of viruses is a critical step in research laboratories and biotechnology industries. Different approaches are commonly applied which either are time consuming (like the plaque and endpoint dilution assays) or do not ensure quantification of only infective particles (like quantitative real-time PCR). In the last decade, a methodology based on the analysis of infected cells by flow cytometry and fluorescence-activated cell sorting (FACS) has been reported as a fast and reliable test for the titration of some viruses. However, this technology needs expensive equipment and expert technicians to operate it. Recently, the "lab on a chip" integrated devices have brought about the miniaturization of this equipment, turning this technology into an affordable and easy-to-use alternative to traditional flow cytometry. In the present study, we have designed a microflow cytometry (μFC) procedure for the quantitation of viruses, using the infectious pancreatic necrosis virus (IPNV) as a model. The optimization of conditions and validation of the method are reported here.

Abstract B056: Modulation of cytokine secretion allows CD4 T cells secreting IL-10 and IL-17 to simultaneously participate in maintaining tolerance and immunity

Abstract Introduction: CD4 T cells secreting IL-10 have been predominantly described as immunosuppressive and include both Tr1 cells and FOXP3+ Treg. In contrast, CD4 T cells secreting IL-17 have been mostly described as helper/effectors (TH17). Recent evidence indicates that some CD4 T cells secreting IL-17 can be of regulatory type, as human FOXP3+ Treg that secrete IL-17 ex vivo while concomitantly exerting suppressive activity have been described. CD4 T cells secreting IL-10 or IL-17 are frequent at mucosal sites where their equilibrium is important for simultaneously maintaining tolerance and immunity to the resident microbiota. Although TH17 and IL-10-secreting CD4 T cells have mostly been considered as distinct populations, the generation of which involves distinct factor, Staphylococcus Aureus specific TH17 clones that co-secrete the two cytokines have been identified (Zielinski,C.E; Nature 2012.484:514-518) underlining the close relationship between the two populations, along with their relevance in host immunity. Their mode of action, however, is as yet incompletely understood. To get insight into these questions, in this study, we addressed the prevalence, characteristics, mode of action and potential role of this type of populations. Methods: We combined ex vivo analysis of CD4 T cells producing IL-10 or/and IL-17 isolated from PBMC of healthy donors with assessment of clonal populations isolated ex vivo using a cytokine catch assay. Results: We found that, in average, 0.9 ± 0.3% and 3 ± 0.9% of memory CD4 T cells secreted IL-10 or IL-17, respectively. The majority of cells secreting IL-10 or IL-17 ex vivo appeared as distinct. However, a subpopulation, representing about 0.1% of the cells, co-secreted the two cytokines. The proportion of FOXP3+ cells was significantly higher among IL-10 single secreting (39 ± 8%) than IL-17 single secreting (13 ± 5%) cells and represented 58 ± 9%, in average, of the cells co-secreting the two cytokines ex vivo. The majority of FOXP3+ Treg within each of the cytokine secreting populations failed to express the transcription factor Helios, suggesting that these cells are peripherally induced (pTreg) rather than Tregs derived from thymus (tTreg). To further assess, we generated IL-10 or IL-17 single secreting and IL-10/IL-17 double secreting populations using the Miltenyi secretion/detection assay and flow cytometry cell sorting. We found that IL-10 secretion is prevalent at early time points after TCR stimulation, independent of co-stimulation and is increased in the presence of the microbial fermentation product butyrate. In contrast, IL-17 secretion is higher at distance from TCR stimulation and in the presence of co-stimulatory signals. Conclusion: These results suggest that the capacity of modulate cytokine secretion depending on conditions might allow IL-10 and IL-17 producing CD4 T cells to maintain tolerance to microbes locally, while retaining the ability to participate in protective immune responses at distant sites. Citation Format: Kanako Saito, Pascale Pignon, Maha Ayyoub, Danila Valmori. Modulation of cytokine secretion allows CD4 T cells secreting IL-10 and IL-17 to simultaneously participate in maintaining tolerance and immunity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B056.

Isolation of Mammalian Oogonial Stem Cells by Antibody-Based Fluorescence-Activated Cell Sorting.

The ability to isolate and subsequently culture mitotically active female germ cells from adult ovaries, referred to as either oogonial stem cells (OSCs) or adult female germline stem cells (aFGSCs), has provided a robust system to study female germ cell development under multiple experimental conditions, and in many species. Flow cytometry or fluorescence-activated cell sorting (FACS) is an integral part of many isolation and characterization protocols. Here, we provide methodological details for antibody-based flow cytometric isolation of OSCs using antibodies specific for external epitopes of the proteins Ddx4 or Ifitm3, alone or in combination with the use of fluorescent reporter mice. Beginning with sample preparation, we provide point-by-point instructions to guide researchers on how to isolate OSCs using flow cytometry.

Anionic/Zwitterionic Lipid-Based Gene Vectors of pDNA.

The use of anionic lipids (ALs) as non-viral gene vectors depicts a promising alternative to cationic lipids (CLs) since they are more biocompatible and present lower levels of phagocytosis by macrophages. Several experimental methods, such as electrophoretic mobility (ζ-potential), gel electrophoresis, small-angle X-ray scattering (SAXS), fluorescence and confocal fluorescence microscopies (FM and CFM), flow assisted cell sorting-flow cytometry (FACS-FCM), and cell viability/cytotoxicity assays can be used for a complete physicochemical and biochemical characterization of lipoplexes formed by an AL, a zwitterionic lipid (ZL), and a plasmid DNA (pDNA), their electrostatic interaction being necessarily mediated by divalent cations, such as Ca(2+). In the present chapter, we summarize the protocols optimized for the mentioned characterization techniques.