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Adipose Tissue Homogenizer & Homogenization Protocol
Abstract The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. Cryopreservation has shown to be most promising but is a fairly complex process, necessitating the use of chemicals called cryoprotective agents CPAs , freezing equipment, and obviously, storage in liquid nitrogen.
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Read Article at publisher's site. While metabolic studies using microdialysis have brought the advantage of studying the lipolytic response of the adipose tissue in situ , it provides only qualitative measures but does not give any information on the contribution of different adipose tissue cell components. Moreover, the number of microdialysis probes that can be used concomitantly in one subject is limited and can be influenced by local blood flow changes and by the molecular size cut-off of the microdialysis probe.
Here we present a protocol to assess adipose tissue functionality ex vivo in AT explants allowing the studies of adipose tissue in its whole context, for several hours. In addition, the isolation of the different cell components to evaluate the cell-specific impact of lipolysis can be performed. We recently used the present protocol and demonstrated that fatty acid release during lipolysis impacts directly on a specific cell subset present in the adipose tissue stroma-vascular compartment.
This assay can be adapted to address other research questions such as the effects of hormones or drugs treatment on the phenotype of the various cell types present in adipose tissue Gao et al. Keywords : Human adipocyte biology, Lipolysis, ex vivo , Explant.
Human white adipose tissue WAT plays a major role in body energy homeostasis. In addition to adipocytes, other cell types are present within human WAT e. The metabolic activity of adipocytes is tightly controlled by the integration of both local and systemic pathways.
Neurohumoral signals modulated in anabolic or catabolic conditions impact on the net adipocyte metabolic activity, i. In post-prandial conditions, non-esterified fatty acids NEFAs originating from the hydrolysis of VLDL and chylomicron particles are taken up by the adipocytes and esterified to glycerol phosphate to form triglycerides packaged into a single lipid vacuole Large et al.
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This process called lipogenesis, is mainly under the control of insulin. In conditions of energy demand such as exercise or fasting, hydrolysis of triacylglycerol through a process called lipolysis results in the release of glycerol and NEFAs into the circulation thereby providing energy to other tissues and organs. Lipolysis involves the sequential hydrolysis of triacylglycerol through the successive action of lipolytic enzymes, i. In human adipocytes, lipolysis is mainly stimulated by catecholamines and atrial natriuretic peptide ANP. It should be noted that in human AT the beta3-adrenergic receptor is almost not expressed and is not functional.
The classical approach to evaluate adipocyte lipolytic responses was developed by Robdell Robdell, who first described mature adipocyte isolation based on flotation after collagenase digestion. Although this technique is used worldwide, it presents several limitations. Firstly, the buoyancy of isolated mature adipocytes will prevent, with increasing time in vitro , their immersion into media and promote direct toxic effects through air contact, ultimately leading to cell damage and disintegration.
Secondly, the isolation process per se alters adipocyte phenotype Ruan et al. Thirdly, the isolated mature adipocytes are disconnected from their natural microenvironment including extracellular matrix and from other cell types vascular cells, immune cells lymphocytes and macrophages and progenitor cells.
Finally, NEFAs originating from in situ lipolysis may have different fates: 1 release into the circulation, 2 re-esterification within the mature adipocytes, 3 potentially taken up by other cell types in the near vicinity of mature adipocytes including progenitor cells. Thus studies on isolated adipocytes do not take into account these different factors.
The present technique allows the study of the lipolytic responsiveness of mature adipocytes for a longer time in their natural context 1 in a closed culture chamber avoiding direct contact with air but with adequate and modulable gas exchange, 2 without the necessity of a collagenase digestion step and 3 in a maintained viable microenvironment. This approach was recently published by our groups and clearly demonstrate that lipolytic stimulation is associated with increased fatty acid uptake by the progenitor cells leading to enhanced adipogenic capacity Gao et al.
Data calculation and analysis were performed using Excel and GraphPad Prism6 software. This approach was recently published by our groups Gao et al.
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A New Personalized Cooling Protocol to Activate Brown Adipose Tissue in Young Adults
A peer-reviewed protocol journal. No publication fee; no access fee. Anonymous reviewer. Pauline Decaunes. Mikael Ryden. Jean Galitzky jean. Original research article A brief version of this protocol appeared in:. Similar Protocols. Reproducibility Feedback. Share your feedback.
Abstract Most studies of human adipose tissue AT metabolism and functionality have been performed in vitro on isolated mature adipocyte or in situ using the microdialysis technique Lafontan, Keywords : Human adipocyte biology, Lipolysis, ex vivo , Explant Background Human white adipose tissue WAT plays a major role in body energy homeostasis. Adipose tissue explant preparation Figure1 Cut adipose tissue into pieces of around cm 3 and put it in a 50 ml conical tubes Figure 1A. Do not exceed 25 ml of AT explant for each 50 ml conical tube this amount is sufficient to perform x Clinicell AT explant Cut with scissors for 2 min Figure 1B.
Centrifuge the tube at x g for 2 min Figure 1C. Remove the infranatant by using a 14 G stainless cannula and a 20 ml syringe Figures H-I. Remove the infranatant by using the 14 G stainless cannula and a 20 ml syringe Figures G-I. Figure 1. Preparation of adipose tissue explants.
AT pieces before mincing; B. First 2 min mincing; C. AT explants after a first mincing, washing and centrifugation; D-F.
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Each photograph shows the aspect of the adipose tissue explant after 2 min mincing with scissors and after ATEM addition and x g centrifugation.
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