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Introduction
Current institution
Publications
Publications (17)
Neural input to the immune system can alter its ability to clear pathogens effectively. Patients suffering mild traumatic brain injury (mTBI) have shown reduced rates of pneumonia and a murine model replicated these findings, with better overall survival of TBI mice compared with sham-injured mice. To further investigate the mechanism of improved h...
Objectives:
Sepsis remains a serious clinical problem despite intensive research efforts and numerous attempts to improve outcome by modifying the inflammatory response. Substance P, the principal ligand for the neurokinin-1 receptor, is a potent proinflammatory mediator that exacerbates inflammatory responses and cardiovascular variables in sepsi...
Sepsis, a leading cause of death in the United States, has poorly understood mechanisms of mortality. To address this, our model of cecal ligation and puncture (CLP) induced sepsis stratifies mice as predicted to Live (Live-P) or Die (Die-P) based on plasma IL-6. Six hours post-CLP, both Live-P and Die-P groups have equivalent peritoneal bacterial...
Objective:
Considerable breakthroughs in the field of sepsis have been made using animal models. Sepsis exhibits a wide array of derangements that may be evaluated in the blood, including the release of proinflammatory and anti-inflammatory cytokines. The Shock journal adheres to the ARRIVE guidelines regarding reporting in vivo results to allow r...
Multiple organ failure in sepsis substantially increases mortality. This study examined if there was greater hepatic, pancreatic, splenic or renal injury in mice that would die during sepsis induced by cecal ligation and puncture (CLP) compared to those who would survive. Mice were stratified into groups predicted to die (Die-P) or predicted to liv...
Accurate measurement of cytokine concentrations is a powerful and essential approach to the study of inflammation. The enzyme-linked immunosorbent assay (ELISA) is a simple, low-cost analytical tool that provides both the specificity and sensitivity required for the study of cytokines in vitro or in vivo. This communication describes a systematic a...
Questions
Questions (13)
I want to label PA with several fluorophores to assay phagocytosis and phagosomal processes. Unfortunately the bacteria aggregate during the labeling process (both live and heat killed @ 80C 30min). I do not have this problem when labeling E.coli.
Method: Grow liquid culture in TSB or scrape from Blood TSA plates. Wash several times in PBS and resuspend to ~ 1010 CFU/mL (same as for e.coli) in PBS pH 7.4. NOTE: resuspending the PA (orange hue) looks like resuspending mucous-like gel.
I then add 10uL of an amine-reactive fluorophore (DCF-SE dissolved in DMSO) and incubate for 1hr room temp. This is done under N2 gas in glass container to limit oxidation of fluorophore. It is during this initial incubation that I notice the PA forms numerous clumps (not a giant clump) that is difficult to disrupt.
If i proceed with the rest of the reaction, it gets worse.
Any ideas on how to prevent the clumping? EDTA or Tween?
I have tried this with both the PAO14 and Boston 41501 strains.
When I isolate Xen41 (Pseudomonas Aeruginosa PAO1 with Luciferase system) from LB/tetracycline plates, place into saline, and read the O.D., the values are not stable and actually decrease. (e.g. 436nm t0=0.333, t5min=.294,t10=.270, t20=.246)
This makes estimating CFU difficult. Why does this happen and how can i work around it?
I have tried resuspending the bacteria in different media (e.g. HBSS, RPMI, LB) and reading at different wavelengths (436, 600, 725, 825nm) but the decrease still occurs.
Any ideas?
