measuring-erythrocyte-complement-receptor-1-using-flow-cytometry

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

University of Reims Champagne-Ardenne

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10(6) tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me2SO without incubation. A cooling rate of ∼1 °C/min to -80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.

Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

The use of inorganic calcium/phosphate supplemented with biopolymers has drawn lots of attention in bone regenerative medicine. While inflammation is required for bone healing, its exacerbation alters tissue regeneration/implants integration. Inspired by bone composition, a friendly automated spray-assisted system was used to build bioactive and osteoinductive calcium phosphate/chitosan/hyaluronic acid substrate (CaP-CHI-HA). Exposing monocytes to CaP-CHI-HA resulted in a secretion of pro-healing VEGF and TGF-β growth factors, TNF-α, MCP-1, IL-6 and IL-8 pro-inflammatory mediators but also IL-10 anti-inflammatory cytokine along with an inflammatory index below 1.5 (versus 2.5 and 7.5 following CaP and LPS stimulation, respectively). Although CD44 hyaluronic acid receptor seems not to be involved in the inflammatory regulation, results suggest a potential role of chemical composition and calcium release from build-up substrates, in affecting the intracellular expression of a calcium-sensing receptor. Herein, our findings indicate a great potential of CaP-CHI-HA in providing required inflammation-healing balance, favorable for bone healing/regeneration.

Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition. Primary HLH occurs early in life due to monogenic biallelic mutations affecting lymphocyte cytotoxicity. Secondary HLH occurs mostly in adults secondary to infections, lymphoma or rheumatic diseases. In this latter setting, lymphocyte cytotoxicity status is not known. We conducted a systematic evaluation of NK cell cytotoxicity in adult patients with secondary HLH. Adult patients with secondary HLH were prospectively studied ex vivo for total lymphocyte count and subtypes, NK cell phenotype, perforin expression and degranulation, natural or ADCC cytotoxicity, in comparison with patients affected by the same underlying diseases without HLH (disease controls, DC) and with healthy controls (HC). Screening for variants of cytotoxity genes was systematically performed. 68 patients were included in the HLH group, 34 in the DC and HC groups, respectively. In HLH patients, a severe and transient lymphopenia, an activated NK cell phenotype (increased CD69, ICAM-1, HLADR, CCR5 expression) and a decreased capacity of interferon gamma production were observed. Mean perforin expression was normal, degranulation tests and NK cell cytotoxicity were not different from DC. A monoallelic variant of uncertain significance affecting one of the lymphocyte cytotoxicity genes, or the perforin variant A91V, was observed in almost 50% of the patients. We detected no major intrinsic cytotoxicity dysfunction in secondary HLH compared to DC and no predicted pathogenic gene variant. The activated NK phenotype profile associated with decreased interferon gamma production appear similar to other hyperinflammatory diseases such as sepsis or systemic juvenile idiopathic arthritis.

Sandra Audonnet

URCACyt,
Flow cytometry technical platform,
URCACyt, Flow cytometry technical platform

University of Reims Champagne-Ardenne

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

Sandra Audonnet

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University of Reims Champagne-Ardenne

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

URCACyt,
Flow cytometry technical platform,
URCACyt, Flow cytometry technical platform