Adipose Tissue - Science topic
Specialized connective tissue composed of fat cells (ADIPOCYTES). It is the site of stored FATS, usually in the form of TRIGLYCERIDES. In mammals, there are two types of adipose tissue, the WHITE FAT and the BROWN FAT. Their relative distributions vary in different species with most adipose tissue being white.
Questions related to Adipose Tissue
In 2019, Volker Bornemann and al, published "Intestinal metabolism and bioaccumulation of sucralose in adipose tissue in the rat" and showed that sucralose could be metabolizable. They found 2 metabolites in urine and feces and found sucralose in adipose tissue.
Nevertheless, no further article published on that point. There is no explanation on the mecanism of the sucralose metabolization.
I have tried to search how sucralose could be changed in the organism but it seems to have no article that answer that question. Does someone have an article on that ?
Can I assess glycogen in adipose tissue using PAS? Has anyone done it? Do you have any reference?
I have read about a relationship between inflammation of adipose tissue and glycogen content in that tissue. I thought about evaluating by PAS. It's possible?
I need to determine the body fat percentage of middle childhood children. But BIA scales won't work in those age group. Kindly suggest me any formula which can work in this age group through skinfold measurements or any other ways.
I have to isolated adipose tissue macrophages from frozen SVF. The following methods can be employed in isolating macrophages as magnetic bead–conjugated antibody cell isolation, density-gradient separation, laser-capture microdissection, and fluorescence-activated cell sorting (FACS). Which of these methods would be best to isolate maximum number of macrophages from frozen SVF?
Out of curiosity, I tested the sensitivity of the DC protein assay to determine how high of a protein concentration you can go without losing sensitivity. The protocol recommends a range between 0-1.5 mg/mL for the protein standards. https://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT448.pdf
I put this to the test utilizing bovine serum albumin (BSA)-derived standards and known concentrations. Concentrations of standards were 0 (blank), 0.25, 0.5, 1 and 1.5 mg/mL followed by known concentrations at 2, 3, 4, 5, 6, 7.5, 10 and 15 mg/mL. BSA was dissolved in water as the buffer. The protocol was precisely followed, samples were assessed in replicate wells.
The DC assay indeed loses sensitivity at 2 mg/mL and this sensitivity drops drastically following this concentration. Something interesting that I noticed was that the standard deviation between replicates also drastically increased >2 mg/mL.
1. My standard curve, which had an R^2 of 0.997.
2. Figure illustrating the divergence between true and measured concentrations
3. Data table showing means and standard deviations of measured concentrations
Based on these data, sticking to a standard curve range between 0-1.5 mg/mL is optimal, and sample aliquots should be diluted to fall below 2 mg/mL for accurate measurement.
1. Cells: 0x-5x
2. Brain and adipose tissue: 0x-5x
3. All other tissue: 10x-20x
I need to facs sort macrophages, there are many surfaces markers which are used for this purpose such as: CD14, CD68,CD64, CD11c, CD204,CD206.
I need to know why certain surface markers are preferred over the others?
We have a primary mesenchymal stem cell culture, obtained from the adipose tissue of a mouse. After about a week, we started seeing these structures that we are not able to identify. We tested twice in PDA agar and the result was negative, there was no growth on the plate. Would really appreciate if you can help us identify and understand what we found in our culture. Most of the pictures are in 20X from an inverted microscope. Thanks.
I have been trying to section frozen adipose tissue samples (high-fat diet and wild type) on a cryostat at -20C at 20um and have not been able to get any sections. The blade doesn't seem to cut through the tissue because there are holes in the sections where the tissue should be. Does anyone have any advice on how to section oily tissue (for H&E)?
The adipose tissue has been fixed in 4% PFA, then left in 15% and subsequently 30% sucrose for a few days before flash freezing in OCT.
The cryostat that I'm using does not go below -20C so I can't lower the temperature further.
Dears, I am doing qpcr of genes with cdna made of RNA from adipose tissue. In the efficiency curve of the primers, the cdna serial dilution does not promote adequate amplification. The less diluted points dots are having less amplification. Example: in the photo, the last amplification is the highest dilution (1:2). I believe it is the presence of an inhibitor. Has anyone dealt with this? Know what to do? The 260/280 and 260/230 ratios are fine. The profile on the electrofluoresis gel is also good. The RNA was extracted with silica glass beads (sterilized and washed with acid), Trizol according to the manufacturer, and then the material was washed with precipitation with 3M sodium acetate and 100% ethanol. Resuspended in nuclease-free milliQ water.
I did a qpcr reaction with SYBR master mix, and cDNA from epididymal adipose tissue. The RNA used for cDNA synthesis was intact (two bands on the electrofluoresis gel), good A260/280 and A260/230 ratios. With the cDNA from adipose tissue it did not amplify none primer. I did it with a liver cDNA and it worked. The tested primers were from genes such as PRDM16, UCP1, Beta Actin. Not even with the one of Beta actin it amplified. Could it be some inhibitor present in RNA or cDNA? Any suggestion?
I have been trying to use the amplex red glucose assay kit (A22189) to measure the glucose in adipose tissue, but when performing it, the standards and all the samples don't show change in the colour, and when analyzing the results, the negative control have an high value of fluorescence. Can someone have an idea what could be happening, or some protocol that I could try?
I would like to cryosect aortic plus perivascular adipose tissue from obese mice. Unfortunately, at -23°C, with a new knife, embedded in Tissue Tek and very careful handling, the tissue is too unstable and it tears when cutting.
Thicker cuts have shown no improvement.
Do you have any tips on how to cut the tissue better?
Also maybe someone who has only cut fat tissue?
I am very glad for help:)
We are having trouble staining mouse brown adipose tissue with this method - it is very faint and nuclei are almost unstained.That would be an hematoxylin issue, so we double-checked every possible item which could be an issue. However, the dye is ok (samples from other tissues are perfect), sample thickness is 4 mm, and every step in the dyeing process is performed correctly - as I said, we aren't experiencing any problems with tissues other than adipose. The samples are fixed in a 4% formaldehyde solution and stored in 70% ethanol.
I'd like to measure NLRP3 inflammasome activation in human adipose tissue sample lysates; specifically by measuring IL-1B and caspase-1 via Western Blot. Does anyone have any recommendations regarding the best antibodies to use for such an experiment? (I see a lot of variation in the literature)
There are many lymph nodes embedded in adipose tissue. When obtaining mouse adipose tissue for flow cytometry, do you need to remove the embedded lymph nodes first, or mix them together to make suspension for flow cytometry? I found that there was a big difference in the number of immune cells between the two processes.
Hello everyone, for a research project, in April we carried out a primary culture of mesenchymal cells from mouse adipose tissue. However, the cells are stressed, the culture doesn't have the morphology that it should have and the proliferation of the cells is not optimal, as they are growing too slowly.
We are using DMEM + HAMF12 culture medium in a 1: 1 ratio, in addition, we add 3.75% of Bobino Fetal Serum (BFS) in order to reduce cell stress.
Could you tell me what else I can do to improve proliferation and reduce cell stress?
I appreciate your advice, thank you very much !!
Right now, my team and I are working on a research project that established a primary culture of mesenchymal stem cells from murine adipose tissue. We executed the establishment in April and, until this day, the cells have experienced low growth rates and difficulties forming colonies and monolayers.
We are using DMEM + HAMF12 as culture medium in a 1:1 ratio, supplemented with 10% of fetal bovine serum and 1% L-glutamine.
We have replaced the culture medium with a fresh one and carried passages when it has been necessary thinking that we would solve this issue, but we have not been successful yet. And, since they have not grown appropriately, we are not able to continue to the next step of the project.
I would appreciate your help and advice. Thank you so much!
Has anyone used these antibodies Bodipy 493/503 - 4,4-DIFLUORO-1,3,5,7,8-PE 10 MG and 4,4-DIFLUORO-1,3,5,7-TETR 5 MG - to stain lipid droplets in fixed cells adipose tissue cells (3T3-L1)? Do you know the difference between them?
Hello, I'm a graduate student with a second semester of master's degree.
What I do in the practice experiment is to separate and culture the SVC from the adipose tissue of the mouse and then FACS.
I'm seeding 5*10^5 each on a 6-well plate.
Cells are well separated and cultured without major problems, and cells are attached well when checked with a microscope just before harvesting.
But there are too few cells after harvesting them.
After the culture, centrifuging and washing with pbs, add 0.5% Trypsin/EDTA 1ml to incubate for 3 minutes. And when I took it out and checked it with a microscope, I found that it was 80 to 90 percent away.
After that, 2ml of cDMEM (10% FBS) was added and pipetteed to harvesting all remaining cells.
Then I turned the centrifuge right away, but I couldn't see the pellet well. I was wondering if it was because the cell was too small, so I made a cell suspension after aspiration and proceeded cell counting, but the harvest rate was only 30%. I'm sure the microscope will check that it's gone well, and after harvesting it, I'll be careful with the aspiration, so I don't know if the cell is going to die, but I can't harvest it.
I'm trying to harvest it in many ways, but I don't know what the problem is...So here's a question for some of the experienced experimenters.
thank you : )
I'm looking for antibodies to stain lipid droplets in fixed cells 3T3-L1 (tissue adipose). Anyone has some suggestions, which antibodies are good for such experiment?
This is an IHC staining for CD142 figure for perivascular adipose tissue. I wonder what are these cells containing particles among mature adipocytes?
Will they be mesenchymal stem cells or immune cells resident in adipose tissues? And what are those particles within these cells?
Hello every one, i´ve been working with rats for a few years but now i'm wondering wether there is a way to calculate the BMI or the adipose tissue in these animals. Also, i've been searching literature about the growth rate in rats, i will be very thankful if you culd help.
I am a PhD student currently looking at adipose tissue (both brown and white) extracted from mice. I extracted the protein using the RELi method and prepped the samples using protein concentrations determined through BCA. When I ran westerns, Ponceau staining indicated inconsistent loading. I then ran the A660 assay on my prepped samples, and found out that the protein concentrations are higher than expected and not consistent across all the samples.
Has anyone else had this issue with adipose tissue samples?
Thanks in advance!
I want to extract acitretine , cis acitretine and etretinate from adipose tissues for analyse with
Can you provide a method for extracting lipophilic components from adipose tissues for me?
It would help me a lot for the sample preparation of this particular tissue since it is the first time I have to extract components from adipose tissue.
I would appreciate any suggestions on methodology to measure catecholamines in frozen adipose tissue. Any guidance is appreciated.
I'm having difficulty understanding my results from doing a ponceau S stain on a nitrocellulose membrane after western blot. I loaded 10ug in lanes 2,4, and 6. 15ug in lanes 3,5,and 7. The 2&3 lanes are goat testes and 4&5 is bovine liver and 6&7 is bovine adipose tissue. I'm more familiar will RNA and EtBr gels and looking for degredation, etc.
The lipid content tends to be problematic for determining protein content and successfully running a gel. Is there a specific lysis buffer recommended or additional steps to break the lipid down?
I am trying to analyse adipose tissue at different time of a surgery. How can I fix the tissue for histological analyse?
PFA seems to take too long to penetrate the tissue to allow a difference in the analysis of 2 sampled at 1 hour interval.
I did not succeed in cutting Direct frozen samples.
Does anyone know what we could do?
How can we harvest more RNA from adipose tissue which contains a lot of fat/lipid? Do we need any special reagent kits? Will we extract more total RNA from adipose tissue of one DIO (diet-induced obesity) mouse than that of one lean control mouse?
I have a GMP grade NB6 collagenasi at 1g.
I would like to use at 0.3 PZ U/mL
How do you suggest to prepare stock solution?
Thank you so much.
i am looking for suggestions for a primary antibody against BrdU. I want to stain adipose tissue sections for immunoflourescence imaging. I tried one primary already without any luck (this specific AB has since been discontinued by the company).
Does anyone have suggestions/experiences with BrdU stainings?
Does anyone know about painful fatty nodules that can be palpated in the lumbar area and can cause cluneal neuropathy, thus low back pain referred to thigh? (They have been named in many ways: episacroiliac lipomas, back mice, lumbar fibrositis, muscular rheumatism)
I extracted these cells from donated adipose tissue as usual protocol: 1mg/ml collagenase for 1h and cell strainer 100 and 70. In culture with DMEM high glucose + 10% FBS. They are in 2 passage in the picture. At day 1 and 2 they present a fibroblast like morphology like seen in the picture but as the days go by (4-5 days a culture) they expand with what looks like cytoplasm prolongations. And they are not dead because when I add trypsin to expand them, changing the dish they go back to the fibroblast like morphology and starts again with the weird morphology.
Hi all, I aim to isolate SVF from the tissue. However, we sometimes receive big chunks of tissue that is not through liposuction. I've been wondering is there a ingenious way to efficiently prep the tissue "chunks" for subsequent enzymatic dissociation? Currently our process involved manually cutting with surgical scissors or other tools, which is very tedious.
I have only had experience with prepping fetal mouse brain with syringes and large gauge needles. I have not test this method on the tissue yet. Does anyone has a tip? Thank you in advance!
Is there any kind of statistical analysis for the comparison of Dietary record such as fried food consumption to compare with the patient's BMI or body fat % or any research paper focussing on this issue? Any help would be useful
I am performing macrophage staining in the oral mucosa using an IFA protocol. There are oval-shaped structures on my scans that have background stain/autoflorescence. After doing some research, I found they are either cross-sections of skeletal muscle or adipose tissue. They sometimes appear ordered and clumped like they are skeletal muscle cross-sections, while other times they are more sporadic as if they would be adipose tissue. I am having difficulty identifying which is which. Do both tissue types usually autofloresce? If so, how can I differentiate between them while looking at my scans? Thank you!
I am looking for knowing the levels of body fat (BF%) estimated by 4 skinfolds (biceps+tricipital+subscapular+ suprailiac) in children aged 6-12y .
I am using QIAamp Mini Kit for DNA extraction and having relatively low yields from heart, skeletal muscle and adipose tissue (around 2 ug). I tried to increase the amount of proteinase K and to extend the incubation time at 56 deg C to get complete tissue digestion but it didn't help much. Any suggestions?
I tried to HE staining white adipose tissue (Wistar rat). However, the membrane has broken, and the appearance is not right. My protocol is similar the found in Methods in Enzymology (MIE): Methods of Adipose Tissue. So could you help me with protocol suggestions?
Hi there! I need advice on a protocol for mouse tissue staining. I will dissect mouse livers and fat pads and look for liver steatosis and adipocyte perimeter. I was planning on doing this with H&E staining, is this the best way to see liver steatosis and adipocyte perimeter?
Once I take out the livers and fat pads, should I put them in 4% PFA for 24 hours and then can I transfer them directly to 70% ethanol for storage for 3 weeks (I am leaving school for winter break and doing the tissue extractions before break and doing histology when I return). Thank you so much in advance!
I have gathered human adipose tissue to assess the association of leptin gene expression with dietary intake of fat. I have measured leptin and GAPDH(housekeeping) gene expression using a real-time PCR devise(qurbet, rotor gene). In this cross-sectional study I do not have a conventional control group to make a deta deta ct.
Would you please advise me how I am able to use the results in order to assess its relation to a quantitative exposure?
Good afternoon everbody!
I would like to ask you a question. I would like to stain specifically my HUVEC that I transplant in the epididymal fat pad of mice. I'm trying to identify in the vascularization the difference between endothelial cells coming from mice vs the HUVECs.
Does anybody has an idea in the best way to specifically identifiy the two population of cells?
Thank you very much for your help
I am looking for a database with abdominal CT scans (lombar 3) with associated segmentation of tissue regions (muscles, bones, viscera and adipose tissue).
I have not been able to find anything online...
Can someone provide me any research article regarding which shows the following;
1. The effect of rs7561317 (22 kb downstream of the TMEM18 gene) on the TMEM18 gene?
2. the risk allele is associated with TMEM18 expression?
3. Does this variant overlap with any known cis-regulatory element?
4. Is there any genotyping and transcriptome for adipose tissue ?
Thank you in advance.
Some white adipose tissue has been stored in -80 degree at least 1 year and I plan to check mitochondria integrity in the adipose tissue. Is that frozen adipose tissue still available for Transmission Electron Microscope study?
I'll appreciate it very much if someone could give me some suggestions!
Reading through the literature, it seems like ~70% of people use Cd11b-conjugated beads, and 30% use F4/80. Does anyone have experience or advice as to which might be better? Are there other cell populations in the stromal vascular fraction that also express one of these markers?
My main priority is recovering a high number of cells, but purity is obviously a concern as well.
I am planning to do orthotopic injection of breast cancer cells (MDA-MB-231 and 4T1 cells) into the mammary fat pad of mice to observe cancer metastasis and tumor growth. In lots of references I see people used growth factor reduced (GFR) matrigel and mixed it with the cells. My question is what is the purpose of using GFR matrigel. Will it makes a different when using matrigel containing growth factor?
As a diabetic exercising 20 hours /week all year round my body fat other than visceral is only 12.5%. I have very high muscle levels but despite strict diet and exercise cannot shift visceral fat.
I know this fat is largely responsible for stopping pancreatic function being normal.
Is there any clear trial results in this area?
I am new to in vivo research. I have tried to induce solid tumors in mice by injecting 4T-1 cells in the mammary fat pad, which is an established procedure. While i mostly ot solid tumors, 2 or 3 of them developed ascites-type tumors with swollen bellies. This may have been due to an accidental intra-peritonial injection. Has anybody noticed this phenomenon before? Also, can anybody please direct me towards any published research along these lines? It will help me a lot. Thanks in advance!
I'm going to do svf from lipoaspirates in my laboratory and I have 500 mg Collagenase NB4 from Nordmark brand. How many dilutions of collagenase do I need for 25 ml of adipose tissue? I did not understand much of the datasheettinde site. Could you please help?
Could you please suggest efficient protocols for isolation and maintenance of mature human adipocytes?
I will isolate adipocytes from adipose tissue obtained after abdominoplasty, all the protocols I researched did not seem consistent, some images obtained left me in doubt as to whether the image obtained was actually from the cell or the fat pockets. Anyone more proficient in the subject could help me? Thank you!
I try to isolate primary adipocyte from mouse epididymal adipose tissue but i always get high debris and RBC. Most of them died after plate the cell. The cells never become confluent. I work along with standard protocol that i attach the link https://bio-protocol.org/e1669 .
It is widely accepted that AQP1, 4, and 9 are expressed in the brain. Among of them, AQP9 belongs to the aquaglyceroporin which is involved in the lipid metabolism.
Given that AQP3, 7, and 9 cross-talk between adipose tissue, muscle, and the liver, I wonder what is the main role of AQP9 in the brain.
Hello colleagues! I'm trying to isolate total RNA from FROZEN samples of adipose tissue. When we extracted the tissue the samples were snap frozen in trizol and few days after I tryed to isolate RNA with Trizol protocol but RNA integrity wasn't optimal.
In the past I isolated RNA from FRESH adipose tissue using the same protocol without any dificult and excelent quality and quantity. I'm sure that samples were preserved in a correct way because we also extracted RNA from other tissues storing them with the same steps, so the tissue is the trouble one.
I alredy add more chloroform in order to separate fat from the other phases but didn't work. Any advice will be helpful! :)
I am planning to measure the adenylate cyclase activity in adipose tissue. I do not want to use radioactivity based methods. Can anyone provide me a simple protocol for adenylate cyclase activity?
I will appreciate if someone could give me a detailed protocol for fixation adn embedding adipose tissue for transmission electron microscopy, in order to see mitochondria cristae.
Thank you very much!!!
What do you think about this statement? Is the deep fatty tissue the CLUE to finally understand unexplained pains. Why it is the deep fatty tissue so neglected? Why it EDEMATIZES so much?
We have strong confidence that our studies will explain FINALLY some unexplained pains.
I would be so thankful if someone could lend us a hand with an issue related to RNA purification.
I'm using the QIAGEN RNeasy Plus Mini Kit for RNA extraction from brain tissue and adipose tissue. I have a set of hipothalami with 1.0ug/ul-1.4ug/ul, and a RIN of 9.4-9.8. But in another set, I got 0.1ug/ul-0.4ug/ul. We should have the RIN of this second set in a couple of days, but I can't get good sleep with this problem.
The manipulation conditions were the same. As soon as the tissue is extracted, it's freezed. The tube contains ceramic beans for later homogeneization with Paracellys tissuelyzer. I always use the same program for tissue disruption. Then I just follow the QIAGEN protocol, with very few adjustments, which I maintain for all the samples. I elute the RNA using 50ul of PCR grade water, and I always incubate the samples 10min before elution at RT.
We need concentrations of at least 0.4ug/ul for qPCR.
Can these concentration difference be due to individual differences? Or is there a flaw in the procedure, significant enough to result in these differences?
Your help and suggestions are much appreciated. Thank you very much in advance!
I would like to perform IF stainings on cryosections of human adipose tissue or fat if you prefer. I have resolved all the problems with cutting sections of fat, but know I have some problems with IF stainings. Could you recommend me some protocols that tackle with IF on fat? Maybe you have some tricks and tips to offer?
I wanted to stain sections for FABP4 and perilipin, but either I have no signal or negative signal all over the section. The same antibodies and protocol worked perfectly fine on cultured adipocytes on plastic.
I am trying to cut 20 um cross sections of murine lymph nodes and small intestines, however, the tissue is fatty and not freezing thoroughly. When I cut the OCT, the cut will have a hole in the center where the tissue should be. Is there a better approach to fatty tissues?
I am taking the OCT directly from the -80C and the cryomicrotome is set to -20C.
We recently published work on the responses of mice to differences in dietary macronutrients in Cell metabolism
After it was published it got a lot of social media attention mostly from non-scientists and mostly along the lines of well OK but its just mice so we can ignore it. My initial reaction was that surely the mouse is a good model of humans. But then I got to thinking well maybe it isn't and the people dismissing the study have a point.
So my question is what are the most significant differences between mice and humans in terms of their adipose tissue metabolism, general control of food intake, and energy balance, that would invalidate (or at least make us question) their use as a model of human energy balance?
Are there examples where mice clearly respond differently to humans to dietary manipulations, or in their metabolic responses to food intake?
Adipose tissue from obese (ob/ob) mice was formalin fixed and paraffin embedded for Opal multiplexed IHC staining. Result revealed aggregated signals, forming vesicles-like bulbs. These bulbs have an average size of 5.2 um. We are not sure about what these aggregated signals would be. We are wondering if they are secretsomes from interstitial cells. Can anyone give us some idea?
I have some human adipose tissue samples. -80 C went down on us over the weekend. We are unsure of how long the freezer was actually down for. When we got it back on the outside thermometer on the door read -6 C. I am worried that these tissue samples are compromised. I am mainly concerned if whether or not it would be ethical to still use these samples in my study. It took a year to collect these samples. Are they totally gone? How can I check their viability?
I am a fresh man in cell molecular biology. I was studying the liver metabolites function and the inflammatory response with exogenous toxicant. I knew the hepatocyte is the metabolite center for human body. It could synthesize a lot of enzymes, albumin and metabolize toxicant. But I was confused whether the hepatocyte could secrete the pro-inflammatory factor, such as TNF-α, iL-6, when liver or hepatocyte was treated with anti-cancer drug doxorubicin (DOX), and exogenous porphyromonas gingivalis. I saw two papers. They reported the upregulation of mRNA in these treatment.
 Zhang W, Yu J, Dong Q, et al. A mutually beneficial relationship between hepatocytes and cardiomyocytes mitigates doxorubicin-induced toxicity[J]. Toxicology letters, 2014, 227(3): 157-163.
Takano M, Sugano N, Mochizuki S, et al. Hepatocytes produce tumor necrosis factor‐α and interleukin‐6 in response to Porphyromonas gingivalis[J]. Journal of periodontal research, 2012, 47(1): 89-94.
Most of other materials reported that the immune cells and adipose tissue was involved the secretion of these pro-inflammatory factor. It seems the function the hepatocyte only working on the metabolites, enzyme synthesis, and energy storage.
Thank you very much!
I have few contaminated RNA samples from adipose tissue which I need to purify. However, treating all samples with purification may minimise the variation but RNA concentration may goes dow as a consequence
We need to cryopreserve small pieces of human adipose tissue (~250mg) in order to cut cryosections for confocal microscopy.
I have RNA samples from adipose tissue of mice , 6 came up with RIN of <3 , from two differently treated groups and different age groups. If I eliminated them, it will go low in power. is there anyway of gene expression analysis of low integrity RNA?