scholarly journals Quantitative analysis of rat adipose tissue cell recovery, and non-fat cell volume, in primary cell cultures

2016 ◽  
Author(s):  
Floriana Rotondo ◽  
María del Mar Romero ◽  
Ana Cecilia Ho-Palma ◽  
Xavier Remesar ◽  
José Antonio Fernández-López ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Lipid Content ◽  
Primary Cultures ◽  
Cell Mass ◽  
Lactate Production ◽  
Tissue Cell ◽  
Cell Recovery ◽  
Fat Cell ◽  
Isolated Cells ◽  
Adult Rats

Background. White adipose tissue (WAT) is a complex, disperse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defence, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT breaking up the matrix collagen fibres, but primary cultures of these cells could not be easily correlated to intact WAT, since often recovery and viability are unknown. Experimental design. Epididymal WAT of 4-6 young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of lactate production. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were also measured. The presence of non-nucleated cells (erythrocytes) was also estimated. Results. Cell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70-75%. Adipocytes were 7%, erythrocytes 68% and other stromal (nucleated cells) 24% of total WAT cells. However, their overall volumes were, 91%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 2.5% of WAT. Conclusions. The methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have found, also, that the "live cell mass" of adipose tissue is very small (about 25 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood). This fact, translates into an extremely high (with respect to the actual "live cytoplasm" size) metabolic activity, which make WAT an even more significant agent in the control of energy metabolism.

scholarly journals Quantitative analysis of rat adipose tissue cell recovery, and non-fat cell volume, in primary cell cultures

2016 ◽  
Author(s):  
Floriana Rotondo ◽  
María del Mar Romero ◽  
Ana Cecilia Ho-Palma ◽  
Xavier Remesar ◽  
José Antonio Fernández-López ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Lipid Content ◽  
Primary Cultures ◽  
Cell Mass ◽  
Lactate Production ◽  
Tissue Cell ◽  
Cell Recovery ◽  
Fat Cell ◽  
Isolated Cells ◽  
Adult Rats

Background. White adipose tissue (WAT) is a complex, disperse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defence, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT breaking up the matrix collagen fibres, but primary cultures of these cells could not be easily correlated to intact WAT, since often recovery and viability are unknown. Experimental design. Epididymal WAT of 4-6 young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of lactate production. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were also measured. The presence of non-nucleated cells (erythrocytes) was also estimated. Results. Cell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70-75%. Adipocytes were 7%, erythrocytes 68% and other stromal (nucleated cells) 24% of total WAT cells. However, their overall volumes were, 91%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 2.5% of WAT. Conclusions. The methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have found, also, that the "live cell mass" of adipose tissue is very small (about 25 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood). This fact, translates into an extremely high (with respect to the actual "live cytoplasm" size) metabolic activity, which make WAT an even more significant agent in the control of energy metabolism.

scholarly journals Quantitative analysis of rat adipose tissue cell recovery, and non-fat cell volume, in primary cell cultures

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2725 ◽  
Author(s):  
Floriana Rotondo ◽  
María del Mar Romero ◽  
Ana Cecilia Ho-Palma ◽  
Xavier Remesar ◽  
José Antonio Fernández-López ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Metabolic Activity ◽  
Lipid Content ◽  
Primary Cultures ◽  
Cell Mass ◽  
Tissue Cell ◽  
Cell Recovery ◽  
Fat Cell ◽  
Isolated Cells ◽  
Adult Rats

BackgroundWhite adipose tissue (WAT) is a complex, diffuse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defense, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT by breaking up the matrix of collagen fibres; however, it is unclear to what extent adipocyte number in primary cultures correlates with their number in intact WAT, since recovery and viability are often unknown.Experimental DesignEpididymal WAT of four young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of glucose uptake and lactate, glycerol and NEFA excretion rates up to 48 h. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were measured. The presence of non-nucleated cells (erythrocytes) was also estimated.ResultsCell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70–75%. Cells showed even higher metabolic activity in the second than in the first day of incubation. Adipocytes were 7%, erythrocytes 66% and other stromal (nucleated cells) 27% of total WAT cells. However, their overall volumes were 90%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 1.3% of WAT.ConclusionsThe methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have also found that the “live cell mass” of adipose tissue is very small: about 13 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood (the rats were killed by exsanguination). These data translate (with respect to the actual “live cytoplasm” size) into an extremely high metabolic activity, which make WAT an even more significant agent in the control of energy metabolism.

scholarly journals Regulation of Na+ transport in brown adipose tissue

1986 ◽  
Vol 235 (2) ◽  
pp. 545-552 ◽  
Author(s):  
K F LaNoue ◽  
C Koch ◽  
D Strzelecka ◽  
T P Kobylski
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Fat Cell ◽  
Isolated Cells ◽  
Intact Cells ◽  
Dependent Atpase ◽  
Brown Adipose ◽  
Active Transport System

In order to test the hypothesis that Na+, K+-ATPase (Na+,K+-dependent ATPase) is involved in the noradrenaline-mediated stimulation of respiration in brown adipose tissue, the effects of noradrenaline on Na+,K+-ATPase in isolated brown-fat-cell membrane vesicles, and on 22Na+ and K+ (86Rb+) fluxes across the membranes of intact isolated cells, were measured. The ouabain-sensitive fraction of the K+-dependent ATPase activity in the isolated membrane-vesicle preparation was small and was not affected by the presence of noradrenaline in the incubation media. The uptake of 86Rb+ into intact hormone-sensitive cells was inhibited by 80% by ouabain, but it was insensitive to the presence of noradrenaline. 22Na+ uptake and efflux measured in the intact cells were 8 times more rapid than the 86Rb+ fluxes and were unaffected by ouabain. This indicated the presence of a separate, more active, transport system for Na+ than the Na+,K+-ATPase. This is likely to be a Na+/Na+ exchange activity under normal aerobic conditions. However, under anaerobic conditions, or conditions simulating anaerobiosis (2 mM-NaCN), the unidirectional uptake of Na+ increased dramatically, while efflux was unaltered.

scholarly journals ПОРІВНЯЛЬНА ХАРАКТЕРИСТИКА ФЕНОТИПОВИХ ЗМІН КУЛЬТУР КЛІТИН ЖИРОВОЇ ТКАНИНИ ТА КІСТКОВОГО МОЗКУ В ПРОЦЕСІ КУЛЬТИВУВАННЯ

2017 ◽  
pp. 113-119 ◽  
Author(s):  
В. В. Ковпак ◽  
О. С. Ковпак
Keyword(s):  
Bone Marrow ◽  
Adipose Tissue ◽  
Cell Cultures ◽  
Bone Marrow Cells ◽  
Primary Cultures ◽  
Tissue Cell ◽  
Adipose Tissue Cell ◽  
Polygonal Shape ◽  
Adipose Tissue Cells ◽  
Number Of Cells

У статті описані дані щодо зміни фенотипу культур клітин жирової тканини (ККЖТ) та кісткового мозку (КККМ) у процесі культивування. Дослідження первинних культур клітин кісткового мозку та жирової тканини щура показали, що вони морфологічно гетерогенні, у їх склад входили: невелика кількість клітин полігональної форми, а основну масу складали фібробластоподібні. За подальшого культивування відмічали процес переходу від гетерогенних культур на нульовому  пасажі до найбільш гомогенних у кінці дослідження. Нами були відмічені відмінності у імунофенотипі культур клітин кісткового мозку та жирової тканини, які не зникали з пасажами. This article describes the changes in phenotype of cultures of adipose tissue cells (ATCC) and bone marrow cells (BMCC) in the process of cultivation. Study of primary cultures of cells of the bone marrow and adipose tissue of rat has shown that they are morphologically heterogeneous, they included: a small number of cells of polygonal shape, and the bulk was fìbroblast-like cells. Process of transition from the heterogeneous cultures at zero passaging to the most homogeneous at the end of the study was noted during further cultivation. We noted differences in immunophenotype of bone marrow and adipose tissue cell cultures that did not disappear with passaging.

Secretion of surfactant protein A from rat type II pneumocytes

1993 ◽  
Vol 265 (6) ◽  
pp. L586-L590 ◽  
Author(s):  
S. A. Rooney ◽  
L. I. Gobran ◽  
T. M. Umstead ◽  
D. S. Phelps
Keyword(s):  
Primary Cultures ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Type Ii ◽  
Isolated Cells ◽  
Adult Rats ◽  
Pharmacological Agents ◽  
Type Ii Pneumocytes ◽  
Phosphatidylcholine Secretion

Secretion of surfactant phosphatidylcholine has been extensively studied and there is evidence that it is a regulated process that can be influenced by a variety of physiological factors and pharmacological agents. In contrast, secretion of the major surfactant protein, surfactant protein A (SP-A), has been investigated to much lesser extent. It is not known whether SP-A secretion is constitutive or regulated and, if regulated, whether its regulation is similar to that of phosphatidylcholine. To address those questions we measured SP-A secretion in primary cultures of type II pneumocytes under conditions identical to those used to study phosphatidylcholine secretion. Freshly isolated cells from adult rats were cultured overnight, washed, and then incubated in fresh medium in the presence and absence of surfactant phospholipid secretagogues. As previously reported for phosphatidylcholine, SP-A secretion was linear with time for up to 4 h. However, the rate of SP-A secretion, approximately 6% of total SP-A (cells+medium) released into the medium per hour, was more than sixfold greater than that of the lipid. Although freshly isolated cells contained 70% more SP-A than cells that were cultured overnight, the rate of SP-A secretion was not significantly different. Secretion of SP-A by freshly isolated or cultured type II cells was not increased by a combination of ATP, terbutaline, the adenosine A2 receptor agonist 5'(N-ethylcarboxyamido)adenosine, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin at concentrations that optimally stimulated phosphatidylcholine secretion. We conclude that secretion of the major lipid and protein components of surfactant are independently regulated.

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