METHODS: 4 color IF for extra-cellular matrix and myosin isoforms

Changes in myosin isoform expression accompanying muscle atrophy during cancer cachexia have been shown (Diffee 2002). For this reason we are very much interested in measuring the cross-sectional area as well as the percentage of muscle fiber populations differing in their physiological properties (e.g. the type of myosin isoform). In the attached immunofluorescence method we highlight the interstitial extra-cellular matrix (collagen 3 + laminin, withe) and three different form of myosin (the fast MHCIIb, green; the slow MHCI,blue; and the intermediate MHCIIa, red). Fibers purely expressing MCH type IIX are black (negative staining). EXTENSOR DIGITORUM LONGUS, top

SOLEUS, bottom

METHODS: Visualisation of myosin isoforms by elecrophoresis and silver stain

Different muscle express different myosin isoforms, for instance the Soleus is enriched in the slow myosin (type 1) while the faster Extensor Digitorum Lungos express the fast myiosins 2A and 2X but not myosin 1. Different isoforms of myosin have different molecular weights and can be separated by electrophoresis and then visualized by silver stain. Attache here is the protocol that Eleonora has learned from excellent tutors who master this technique. If interested in myosin isoform analysis, be patient and follow the link.

Figure legend. Myosin isoforms from the Extensor Digitorum Longus (EDL, first two lines) and from the Soleus (SO, lines 3 and 4) murine muscles. The isoforms 2A, 2X have the same apparent weight, the isoform B is specific pf the EDL while the isoform 1 characterizes the SO.

METHODS: Murine muscle dissection from the hinlimb

Here is the link to our illustrated,step by step method for dissecting several skeletal muscles from the hindlimb of a mouse.

EXPERIMENTAL MODELS: BALB/c substrains & running behavior

All BALB/c mice are not equal. In spite of being an inbred strain, there are several SUBstrains that diverged decades ago.In the attached notes, I summarized the names and origins of the the three main BLAB/c substrain, i.e. the Balb/c AnNCrl, the Balb/c J and the Balb/c ByJ mice. They are sold by Charles River, Jackson Laboratories and other vendors, often depending on geographical localization (CRL breeds and sells different substrain in different countries). Concerning the propensity to voluntary running (on a wheel) I could not find any information on the CRL Balb/c AnNCrl substrain,and our recently published results are probably among the first to be released on this substrain. On the contrary,a bibliographic search pinpointed strinking differences between sex, and between different BALB/c substrains, for what concerns running activity (Lightfoot et al. J Appl Physiol. 2010 September; 109(3): 623–634). The infos are summarized here. The source of the data on rod and wheel running activity come from a wonderful database, available on the Jackson website (http://phenome.jax.org/), reporting all major phenotypes of many different mouse strains. Additional data are in the post "EXPERIMENTAL MODELS: wheel running"

Grip stenght test

Here is provided the link to our standard method for measuring the force of a mouse. This method is based on the measure of the grip force by a dynamometer, while the mouse is being pulled by its tail.

Indo-Italian Forum on Biomaterials and Tissue Engineering

A new space for scientific collaboration, exchange of human resources and grant rising was born in New Delhi last week: the Indo-Italina Forum on Biomaterials and Tissue Engineering. During the APA INternational congress on Advances in Human Healthcare Systems (Healthcare India 2012) we participated to the Indo-Italina Symposium on Tissue Engineering, where several aspects of cell interaction with biomaterials were addressed. In this context the Forum was born, which we hope will become a catalyzer for further exchanges between the two countries. More details on the newborn Indo-Italian Biomaterials and Tissue Engineering Forum (i2bite) have been reported on the ENEA newsletter (article in Italian). The link to the Forum web pages (under construction) is here. More details on the event (in Italian) are here.

Candidate for ISAC councilor

I am candidate as ISAC councilor. The elections are in the next few weeks (vote end by March the 30th). Are you an ISAC memeber? Please, vote. All the instructions on how to vote will be soon linked here. Not an ISAC member? Do you want to know more about ISAC? Please, click here to know more about the the International Society for Advancement of Cytometry. Below, there are a few notes on my biography and thoughts about the society. Biography Born in Latina (Italy), I performed both my undergraduate (Biological Sciences, summa cum laude, 1995) and graduate studies (Doctoral degree in Cell Science and Morphogenesis, top mention, 2000) at the Sapienza University of Rome. Since then I accumulated a quite varied experience abroad, as a visiting scholar at the Stanford University (Stanford, CA; 1999), then as a postdoctoral fellow at the Mount Sinai School of Medicine (New York, NY; 2000-2003), as invited researcher at the Myology Group, UMR S 787 Inserm, UPMC (Paris, FR; 2007), where I ultimately returned in 2010 as a Maitre de Conferences, i.e. assistant professor, at the University Paris VI/Pierre et Marie Curie. I also held in Italy the responsibility of the Laboratory of Electron Microscopy and Calcium Imaging (Rome, IT; 2004-2010). All this was a lot of fun, since I could feed myself with great science - not to mention outstanding culinary experiences - from very different environments. For all the above, I have to acknowledge several mentors, including Laura Teodori and Sergio Adamo in Rome, Marco Conti in Stanford and David Sassoon in New York. For all the details, please view my full CV, while works in progress can be followed through my blog. As a cell biologist, I dealt with analytical cytology quite early in my career. Whilst not being exclusively specialized in flow cytometry, I exploit the incredible power and the elegant performance of flow cytometry analysis to address several questions related to my scientific interests. I am mostly interested in the control of skeletal muscle differentiation and homeostasis and, more recently, in tissue engineering applications for regenerative medicine of this tissue. I had the honor to become an ISAC Scholar in 2006 and since them I am member of this society. Interests and vision I came across ISAC through my mentor, Dr. Laura Teodori at a time when I was doing my postdoctoral training about 10 years ago. Being scientifically seduced by the powerful applications of analytical cytology I started to attend ISAC international congresses and to participate more actively to the initiatives of the Society. At the XXIII ISAC congress in Quebec in 2006, I was awarded the ISAC Scholarship. As an ISAC Scholar I was encouraged to collaborate to the educational and organization strategies of the Society. I was young, mobile and without a tenured position. I felt sympathetic with the younger members of the Society and concerned about the typical issues they have to deal with: practicing, traveling, finding the resources to do that. By co-chairing a subcommittee of the MSC (Membership Services Committee) dedicated to Students' services in 2004-'08, I helped with the divulgation of skills and resources aimed to improve student members' success rate when applying to mobility grants. For instance, at the XXIV ISAC congress in Budapest in 2008 I participated to the Scientific Professional Skills Workshop with a presentation entitled “Short term mobility grants: tips and hints.” These issues found concrete development in the ISAC web page highlighting grants opportunities for short term mobility and for young fellows (originally published on the ISAC web site, http://www.isac-net.org/content/view/693/137/). I was responsible for that page, updating it twice a year and offering tutoring and advice to ISAC student members, with the aim to help their mobility and grant rising capacity. Today, from a more mature position inside the Society I wish to contribute to consolidate ISAC strengths and to further develop its potentials, hence my interest in the candidature for an ISAC Councilor position. In this position I could possibly exploit my growing experience and creative attitude to serve our common goals. I foresee two critical issues ISAC shall deal with in the incoming years: 1) geographical and intergenerational growth and 2) interaction with novel scientific and technological research areas. 1) I am convinced that our Society should invest more than ever on youngs. As far as I know ISAC educational and scholarship programs see an unprecedented success, which highlights the interests into our society by young researchers. ISAC should be even more attractive than today for them. In order to do so, we should pursue our politic of open access for young members, and of tutoring and education initiatives. Also, ISAC could set up initiatives aimed to assist its younger members in fund rising (startups, mobility). The initiatives could range from helping members to find senior partners for big grant applications to assisting members to identify calls and apply to them (a task often performed by specific services that are present only in major departments and universities). Obviously, the current programs dedicated to tutoring and visiting initiatives for young members would be synergistic with the novel actions I propose. An international society such as ISAC should become the catalyzer for exchanges and interactions not only vertically (between generations) but also horizontally (between emerging countries, where it is largely underrepresented, and consolidated scientific environments). So I would love to see novel initiatives, aimed at networking and diffusing the analytical cytology, especially targeted to the younger members and to researchers from emerging countries. 2) While being well developed in the US, tissue engineering and regenerative medicine are novel, fast growing disciplines in several countries, including european countries such as Italy and France, or Asian giants such as India and China. This area of Medicine is attracting more and more public and private financing, given its translational nature and high technological content, which in turns stimulates a growing involvement by scientists. Given the foreseen rapid shift to clinical practice in this field (indeed a reality for certain applications) it is of pivotal importance to set up at the same time innovative approaches and safety/quality control procedures for stem cell isolation and transfer, as well as for immunological stereotyping of host-implant interactions. In this context there is an important opportunity for ISAC to become the reference for such procedures and approaches. Thus, I would like to establish initiatives aimed to boost the collaboration between ISAC and non-ISAC members for regenerative medicine applications. An other level of interaction could be within the journals associated to those societies which represent the analogues of our Cytometry; politics encouraging cross-publication (and even cross-advertising for the scientific societies) could be built and I would be happy to collaborate on this. I wish that the publication of this text can be a matter of discussion and engagement by others and myself independently from my candidature as ISAC councilor. I really think that these issues are relevant for ISAC development and I am looking forward to seeing them dealt with.

Quencing autofluorescence

Method for quencing background fluorescence due to aldehydes or autofluorescence