Science topic

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.
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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 ?
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Sucralose is a synthetic sweetener that is approximately 600 times sweeter than sucrose. It is made by partial chlorination (replacing OH groups with Cl) of sucrose. Sucralose is very heat stable and can be obtained in powdered form for use in baking applications. It has numerous food applications, including candy, soft drinks, and sweet syrups. Sucralose is available in granulated and powdered form. Granulated sucralose is mixed with fillers to provide a measure for measure substitution with table sugar (sucrose). The powdered form of sucralose contains 90% bulking agents such as maltodextrin, that are a metabolizable form of carbohydrate. A 50/50 mixture of sucrose and sucralose, plus a bulking agent, is available for baking applications. This mixture reduces caloric content and enables browning reactions in baking applications. The safety of sucralose has been extensively investigated and is considered safe by FDA, the Joint FAO/WHO Expert Committee Report on Food Additives, and the European Union's Scientific Committee on Food. The ADI for sucralose is 5 mg per kg of body weight which equates to approximately 390 mg for a 175 lb person.
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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?
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Check this article: They worked with PAS on embryonic adipose tissue cryosections.
However, PAS is not specific for glycogen, since it stain other molucules too, as stated here
Good luck!
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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.
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Greetings!
You can find the required formula for fat mass percentage and fat mass index based on BMI, age and sex on the following link:
Also, in supplementary materials, you can find Excel table with calculations, you only need to enter your numbers.
Hope it is helpful.
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Do you have an idea of the best way to measure body fat percentage and determine whether a person is fat or not?
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There are several methods that can be used to measure body fat percentage and determine whether a person is within a healthy range. Some of the most common methods include:
  1. Skinfold thickness measurement: This method involves using calipers to measure the thickness of a pinch of skin and fat at specific points on the body. The measurements are then used to calculate body fat percentage using a formula or chart.
  2. Bioelectrical impedance analysis (BIA): This method involves sending a low-level electrical current through the body to measure the resistance of tissues to the current. The resistance is used to calculate body fat percentage.
  3. Dual-energy x-ray absorptiometry (DXA): This method uses x-rays to measure the composition of the body, including fat, muscle, and bone. It is considered to be one of the most accurate methods of measuring body fat percentage, but it is also more expensive and exposes the person to a small amount of radiation.
  4. Hydrostatic weighing: This method involves weighing a person underwater and using the difference in weight to calculate body fat percentage. It is considered to be one of the most accurate methods of measuring body fat percentage, but it can be inconvenient and uncomfortable for some people.
It is important to note that no single method is considered to be the "gold standard" for measuring body fat percentage, and different methods may produce slightly different results. Therefore, it is generally recommended to use multiple methods in combination to get a more accurate assessment of body fat percentage.
To determine whether a person is within a healthy range of body fat percentage, it is generally recommended to use guidelines from organizations such as the World Health Organization (WHO) or the American Council on Exercise (ACE). These guidelines provide ranges for different age and gender groups, and can help to identify whether a person is at an increased risk for health problems due to excess body fat.
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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?
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Hello Syeda Ali
Choosing an appropriate technique depends largely on the required purity, yield, and speed, as well as on cell viability and functionality after extraction.
Density gradient centrifugation has limited throughput and specificity. The two most common methods used are MACS and FACS which improve on these parameters in different ways.
The main difference between MACS and FACS is that MACS provides bulk separation of cells, whereas FACS is based on single-cell sorting. Also, MACS has a higher throughput of the two techniques, while FACS utilizes more parameters and, thus, is more specific.
So, from the techniques that you have mentioned, I would suggest you choose either magnetic bead–conjugated antibody cell isolation or FACS.
Best.
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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.
Attached files:
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.
Recommended dilutions:
1. Cells: 0x-5x
2. Brain and adipose tissue: 0x-5x
3. All other tissue: 10x-20x
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Indeed, that is what I'm saying Sebastian, the color intensity changes at high concentrations are no longer proportional to changes in protein. This does represent a loss of sensitivity by definition at concentrations >2 mg/mL
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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?
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Hello Syeda Ali
Macrophages can be categorized into two main types: M1 and M2 macrophages. The M1 type, referred to as classically-activated macrophages, are activated by pathogen invasion and play a large role in the immune response to foreign pathogens such as bacteria.
The M2 type, referred to as alternatively-activated macrophages, play a role in wound healing and tissue repair, and have an anti-inflammatory role.
M2 macrophages can be further divided into M2a, M2b, M2c, and M2d subcategories. These macrophages differ in their cell surface markers, secreted cytokines, and biological functions.
So, depending upon the type of macrophage you can select the markers.
For instance,
CD68 and CD11b are total markers of macrophages.
M1 and M2 macrophages have specific markers.
M1 Macrophage Marker
For M1 you may choose CD80, CD86, CD64, CD16 and CD32 as markers. In addition, the expression of nitric oxide synthase (iNOS) in M1 can also serve as phenotypic marker.
M2 Macrophage Marker
CD163 and CD206 are major markers for the identification of M2 macrophages. Related surface markers for M2-type cells also contain CD68. Compared with marker CD68, CD163 is more selective to macrophages, so CD163 can be used as a highly specific marker for M2-type macrophages.
M2 macrophages are sub grouped into M2a, M2b, M2c, and M2d.
M2a macrophages
M2a are activated by IL-4 or IL-13. M2a macrophages lead to the increased expression of IL-10, TGF-β, CCL17, CCL18, and CCL22. These macrophages enhance the endocytic activity, promote cell growth and tissue repairing.
M2b macrophages
M2b are activated by immune complex, Toll-like receptor (TLR) ligands and IL-1 receptor ligands, and they can express IL-6, IL-10, TNF-α, and IL-1β. Based on the expression profiles of cytokines and chemokines, M2b macrophages regulate the breadth and depth of immune responses and inflammatory reactions.
M2c macrophages
M2c also known as inactivated macrophages, are induced by glucocorticoids, IL-10 and TGF-β. These cells secrete IL-10, TGF-β, CCL16, and CCL18 and play crucial roles in the phagocytosis of apoptotic cells process.
M2d macrophages
M2d can be induced by the TLR antagonists, and M2d macrophages express IL-10, IL-12, TGF-beta and TNF-α.
Best.
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Hi everyone!
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.
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This looks ECM (top picture). Since you 've ruled out potential contamination, the other structures are likely medium components that precipitate and aggregate for some reason..
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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.
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You should make the Frozen room colder to -30 because of high concentration of Lipid and also you can cut your tissues with thicker trims like 8 mm, however, some other ways like embedding your tissues with Cold Nitrogen instead of Frozen glue would be more suitable.
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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.
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I find a lot of people don't realise quite how dilute cDNA can afford to be.
cDNA synthesis buffer itself is inhibitory to PCR (it contains DTT, for example), and you can also inhibit your reaction by saturating the system with too much target (or too much non-specific target).
In almost all cases, cDNA should be diluted prior to qPCR.
I typically dilute all my cDNA 1/20 (so for a 20ul prep I add 380ul water, giving me 400ul of final cDNA), and using 2ul of this per reaction is _plenty_: it allows me to reliably detect even low abundance genes.
For reference I use ~1600ng of RNA in a 20ul cDNA synthesis reaction, so 2ul of diluted stock is ~8ng of cDNA, assuming 1:1 conversion.
In your case it may be nothing to do with the lipids and everything to do with just...generally using too much cDNA.
When running dilution curves of cDNA historically, I expect the extremes at either end to be unreliable: with too much cDNA you have all the issues noted above, and with too little cDNA you have stochastic effects and thus highly variable data. This is FINE. The whole point of a dilution series of cDNA is to establish the range over which your reaction is linear and trustworthy.
Find that range, use it, don't worry about the extremes.
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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?
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How about your positive and negative control? Is that valid?
If Beta-actin also didn't amplified, it is either there is inhibitor in the master mix or samples or there is no cDNA inside the sample.
Beware of ethanol or alcohol, which can serve as the source of inhibition. If your positive also without amplification, it might be the problem of master mix or qPCR protocol.
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Hello,
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?
Thank you
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Thanks for the info. Glucose is metabolized immediately, so you should look at the 2-DG uptake.
This method is easiest and most reliable using RI labeled 2-DG, but colorimetric and chemiluminescence detection is also available.
(However, I only have experience with RI-based method.)
*Colorimetric method
*Chemiluminescence
Glucose Uptake-Glo™ Assay
Please carefully examine the methods.
Good luck!
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I want to dissect inguinal white fat in mouse, but I don't know where exactly it is. And I am confused whether posterior subcutaneous adipose tissue is the same with inguinal adipose tissue.
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perturbations and that body fat distribution is an important determinant of obesity-related complications. Individuals with increased upper-body adiposity are disproportionately burdened by obesity-related diseases, compared to lower-body obese individuals. Thus, it is paramount that studies continue to elucidate the pathways linking various adipose pads and depots in relation to health and disease, as well as the mechanistic underpinnings dictating how body fat is distributed in order to answer fundamental questions. Rodents are commonly used to model features of human metabolism and obesity, yet it is unclear to what extent rodent fat pads are a suitable model of human fat depots. Here, we have highlighted examples of both shared and divergent traits among rodent fat pads and human fat depots. Given some of the stark differences in adipose tissue location and function among species, we urge careful consideration in experimental design and interpretation when attempting to draw definitive parallels between rodent fat pads and human fat depots.
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Dear colleagues,
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:)
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Thanks for the tip. Until now, I have not fixed the tissue in succrose before embedding.
I will try that and also cut in colder temperatures:)
Kind regards
Jasper
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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.
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Dear Parhesh Kumar , I haven't personally tried it, but my guess is that is should be possible, since cryofixation methods are applicable to adipose tissue. You can maybe if someone has conducted it even if it was with a different tissue to check whether they performed the following steps with different staining conditions. Good luck!
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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)
Thank you!
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Hi, arigo has an NLRP3 Inflammasome Antibody Panel which includs antibodies to NALP3, ASC, and caspase1 with small package. Please refer to the detail at https://www.arigobio.com/NLRP3-Inflammasome-Antibody-Panel-ARG30331.html.
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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.
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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 !!
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Hi Archana, yes, our medium was already supplemented with L-glutamine sodium pyruvate, but still, the cells remained stressed and fragile. In fact, there are very few in the culture bottle now and we don't know if we will be able to save it.
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Hello everybody,
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!
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Years ago, before MSC were identified, I used to culture murine bone marrow stromal cells. In that culture system, the medium was RPMI 1640 + 5% FBS - NOT heat inactivated. Since then, I have worked with human MSC from bone marrow using the spicule fraction, and for that work, I used DMEM - low glucose/ 1 gm per liter + 10% FBS (also not heat inactivated).
Some key points I have found are 1) seeding density is important - at least for bone marrow stromal cells. They produce an autocrine factor [which turns out to be M-CSF] and at too low a cell density, they do not proliferate.
2) Passage before the culture reaches confluence is crucial - particularly for MSC. MSC undergo contact inhibition - a major decrease in gene expression - and once they are in that state, they grow painfully slowly. This is likely where bFGF comes in - as noted by Wondong Yu.
3) Use FBS that has not been heat-inactivated. The grade of the FBS seems to be important - not all products work for MSC cultures. You need to test suppliers. We have had good results with FBS from Sigma - catalog # F2442.
4) Using low glucose medium is important - at high glucose, you get an outgrowth of adipocytes. DMEM comes at either 1 gm per liter or 4.5 gm per liter. Use the 1 gm/liter DMEM.
One other point to note - DMEM/Ham's F12 at 1:1 is essentially the same as Iscove's Modified Dulbecco's Medium {IMDM].
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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?
Thank you,
Ana
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These fluorescent lipophilic dyes, which partition into the non-polar lipid droplet core, are useful lipid droplet markers. BODIPY™ 493/503 4,4-DIFLUORO-1,3,5,7,8-PE 10 MG is according to: https://www.thermofisher.com/order/catalog/product/D3922#/D3922A dye (so not an antibody) that is described as ”With its nonpolar structure and long-wavelength absorption and fluorescence, BODIPY® 493/503 can be used as a stain for neutral lipids and as a tracer for oil and other nonpolar lipids”.
4,4-DIFLUORO-1,3,5,7-TETR 5 MG (BODIPY™ 505/515 (4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene)) https://www.thermofisher.com/order/catalog/product/D3921#/D3921is described as ”With its nonpolar structure and long-wavelength absorption and fluorescence, BODIPY® 505/515 can be used as a stain for neutral lipids and as a tracer for oil and other nonpolar lipids.” Indeed, the same. Looking at the structures the difference is one methyl group.
Perhaps the following papers is of interest to you, it discusses the (possible) advantages in terms of spectral features between different dyes:
Best regards.
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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 : )
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Hi Kim,
I am not working on SVF cells. But, reading your complaint about low cell yield, I guess the reason might be a simple issue. Here are some suggestions:
1. Make sure you grow enough number of cells. 6-well has a small surface area, and you may get quite low yield in the end. If cell size is relatively large, they may look confluent under microscope, but the number might be much smaller than you expect when you count them.
2. Make sure majority of cells detach from plate after digestion with trypsin. You can adjust time to ensure maximum cell detachment but no more than 5min.
3. Centrifuge cells at a slightly higher speed or longer at your original speed. This may increase your cell yield in case some cells didn’t precipitate well.
4. If you are using fixed-angled centrifuge rotor, your cells may stick to tube walls and you may loose them during suction. If that’s the case, you can switch to swinging rotor.
5. When you have low cell yield, you should be extra careful not come too close to pellet with suction tip. Just leave some media above the pellet.
These would possibly increase your cell yield. Good luck!
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Hello,
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?
Thank you,
Ana
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Hi Anna, are you trying to label the lipid, or the proteins sequestering the lipids? For labeling mature droplets, antibodies targetimg perilipin would work. For labeling (or imaging) the neutral lipid, bodipy can be used.
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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?
Many thanks!
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well these are adipocytes for sure !
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Hi everyone, I am wondering if someone has a good protocol and antibodies for M1 and M2 macrophage staining by immunohistochemistry of murine adipose tissue?
Appreciate your advice.
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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.
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Hi everyone,
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!
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There is a number of issues that may occur with your measurements. Uneven loading due to incorrect measurements of total protein is a common problem for every researcher using WB. Quick solution for your experiment is to quantify the bands using image J, calculate the percentage, and then use it as the correcting factor for your initial loading values (i.e if sample 1 is 100%, but sample 2: 150% multiply sample 2 total loading to 0.5) and rerun the gel. In terms of fixing your quantification process, it is hard to say what exactly may be wrong as there are many factors, try to use update your standard curves.
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I want to extract acitretine , cis acitretine and etretinate from adipose tissues for analyse with
ESI-LCMSMS.
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.
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These drugs belong to the chemical class of retinoids, so I suggest you to consult scientific literature, you will find many protocols for extraction/purification of retinoids and vit A-related compounds from adipose tissues (e.g. https://doi.org/10.1002/neu.20243; https://doi.org/10.1016/S0003-2697(02)00662-0 ).
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I would appreciate any suggestions on methodology to measure catecholamines in frozen adipose tissue. Any guidance is appreciated.
Best,
Molly
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You can try a liquid-liquid extration. Make sure your aqueous phase is acidic (Perchloric acid pH 1.0) and I also like to deoxygenate before with nitrogen stream. After the extraction, you can perform either direct Electrochemical measurements or HPLC-EC or HPLC-UV.
Good luck!
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Greetings,
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.
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Hi, maybe you have resolve your problem but I think you have to improve some gel condition, and liver samples preparation.
Did you make coomassie on gel, out of curiosity?
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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?
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The best and only adipose tissue protein extraction kit is the Minute™ Total Protein Extraction Kit for Adipose Tissues/Cultured Adipocytes. It uses a strong cell lysis buffer and a patented spin column filter technology. Results give a complete protein profile and high yield unlike RIPA buffer. https://inventbiotech.com/products/minute-total-protein-extraction-kit-for-adipose-tissues-cultured-adipocytes?_pos=1&_sid=4dc367ec1&_ss=r
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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?
Thanks
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You should rather use 10% neutral buffered formalin as a fixative. For effective and early penetration in tissues fixative can be directly injected through vascular system so that it reaches tissue fast and fixation starts immediately. Even thereafter when you collect and keep tissue in fixative it needs minimum 24-48 hrs to get fixed properly (it depends on thickness of tissue collected)
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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?
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Hi Monika,
Maybe it's too late you already have the answer but I experience it also and I was surprised! :) I believe it can also help other people in the future :) When you try to isolate total RNA from the adipose tissue I recommend you to remove fat layer after homogenization. Normally, if you experienced 4 phase after chloroform it means that you used quite big amount of tissue and there was a lot of fat layer. So, it is important to remove fat layer by pipet as much as you can before adding chloroform. To achieve this separation correctly, you should do a centrifugation right after homogenization process. 2-3 mins at max speed. I'm using 200ul tips to remove fat layer and it is working so well! Rest of the protocol same than regular RNA isolation procedure.
Good luck Monika Nanda and others.. :)
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Dear all,
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.
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It depends on the collagenase activity reported by the manufacturer.
If for example the activity was ≥ 0.18 U/mg and you wanted a 0.3 U/mL stock the calculation would be:
(1000mg) X (0.18 U/mg) / (0.3 U/mL) = 600 mL
This stock can be made in in all buffers which are commonly used for cell isolation.
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Hi everyone,
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?
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Thank you very much, i will try that!
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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)
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Hello Luca,
The nerve impingement could be done -among other reasons- by the fatty tissue embedded within the lumbar fascia. Little is known about the structural changes that the deep fatty tissue can undergo. For exemple, local hyperplasia of the deep fatty tissue could cause cluneal neuropathy. Local hyperplasia palpable as nodules in the lumbar region have been called "back mice" and have been related to chronic low back pain.
Thanks,
Marta
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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.
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Hi Jiulia, from your pics it looks like you have a heterogeneous population with senescent cells. But they do have fibroblast-like morphology! I would add 10-20ng/ml of FGF-2 to help... Anyway you should obtain a homogeneous population after 2-3 passages in culture. Which passage are you cells in the figures?
Best,
Yuri
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I need to fix the Adipose tissue from Mice for IHC. I am looking for a good protocol to follow.
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Good day,
As other tissues. Fix for 24-48 h in 10% buffered solution of formaldehyde, then make parafin embibition by standart protocol with isopropanol or ethyl alcohol, and then get parafin blocks. It is in brief. Specific preparation of adipose tissue needed in cases of special stains like sudan III or kongo rot, when you need to visualise the fats.
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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!
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Hi,
you can try gentleMACS™ from Myltenyi Biotec.
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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
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I like to add my view in respect of the answer given by Rasha Shakir Nima.
It is not true to suppose that any given pair of variables are linearly related so that any given set of data on a pair of variables can be represented by linear regression line.
Therefore , the degree of regression for the given set of data is to be determined first. This can be determined by testing the significance of regression coefficients (starting from linear regression coefficient to the regression coefficients of higher order).
After determining the degree of regression, the corresponding regression analysis is required to be performed.
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For instance if the body fat cut off is based on some western standards, how can we determine cut off for our study, based on the study results? ROC curve?
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If your data are distributed in a Gaussian way (normal distribution), then the cut off will be the mean+2SD. However, and to be more precise, you can create a 2*2 table, from which you can calculate the performance indicators of your test- mainly the sensitivity and specificity- from which you can construct a Receiver Operator Characteristic (ROC) curve where your tests' cut off shall be the point which possess, theoretically, the maximum sensitivity and specificity values.
Best wishes
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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!
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hi paper,
you may coupling your macrophage staining with anti myosin heavy chain or perilipin immunostaining to mark fibers or adipocytes, respectively. Alternatively, you can perform a hematoxylin and eosin stain. In this case, adipocytes appear as white vacuolar unstained cells while fibers will be stained in red
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I am looking for knowing the levels of body fat (BF%) estimated by 4 skinfolds (biceps+tricipital+subscapular+ suprailiac) in children aged 6-12y .
Thanks!
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Dear Alicia Bibiana,
There is a reference developed in 2006 by the Epinut research group from the Complutense University of Madrid but it is based on Spanish children. It provides percentiles by sex and age based on the 4 basic skinfolds and the equations from Durning & Womersley, Siri, and Slaughter. Here is the link to the article: https://www.analesdepediatria.org/es-pdf-13090892
Besides, there is a classical classification proposed by Westrate and Deuremberg in 1989 in where they stated that the presence of obesity in prepubescent children exists when the %body fat obtained by anthropometry is greater than 30% without distinction of age. This is the link to the article: https://academic.oup.com/ajcn/article-abstract/50/5/1104/4695440
I hope this could be useful for you. If anyone knows any other reference I'd also appreciate reading it.
Best wishes!
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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?
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Jana Tumova Thanks Jana!
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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?
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Hi Joao, The adipose tissue is very delicate and sections are difficult to stretch without damaging them. I would suggest that you prepare a 10% alcohol (ethanol or IMS) in distilled water and pipette a large droplet of this solution on a positively charged slide (if you wish to collect serial sections - prepare a row of such slides). Place the adipose tissue section on the surface of the droplet. The surface tension of the droplet unfurls creases of the section and that allows for the next step - careful lowering of the slide into a water-bath (keep the temperature above 45 deg C, but you need to experiment to find the best temperature which gives you enough time for collecting the section without rushing (depends on your thermostat and the type of wax you are using). Watch the wax becoming more translucent and immediately collect it on the slide.
Until you are used to this method - it would be better to collect one section at a time as it is easy to miss the best moment for collect a section from the water-bath.
Good luck with your project.
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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!
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C. Juneja Chloe Henderson
Best variant is to fix tissue in big volume (over 20-25 volumes of tissue) of 4% phosfate buffer formalin (pH over 7.4) for 24-48 hours and then do histological embibition of tissues. Better leave parafin blocks before winter breaks.
Ethanol for fixation is not a good idea especially for a long period. Ethanol dries tissues and H/E stained tissues will be with artifacts.
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Dear colleagues
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?
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Dear Dr/ Lee
I appreciate your reply. Would you please provide me a reference for 2^-dCT?
Thanks,
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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
Charles
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I did a similar thing in the past, we transplanted stem cells tagged with dapi (there is a version sterile for cells) and we stained the cells we put inside, the only draw back is that fades after a few days because the cells replicate and the dna (stained) becomes half at every reproduction cycle. If is for a short time you will be able to use this technique, again you can also use iodine red for the same purpose
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hello,
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...
thank you
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TCIA Collections: TCIA is a service which de-identifies and hosts a large archive of medical images of cancer accessible for public download. The data are organized as “collections”; typically patients’ imaging related by a common disease (e.g. lung cancer), image modality or type (MRI, CT, digital histopathology, etc) or research focus. DICOM is the primary file format used by TCIA for radiology imaging. Supporting data related to the images such as patient outcomes, treatment details, genomics and expert analyses are also provided when available.
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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.
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Hi Adil
this SNP is far away the 5'UTR of the TMEM18 gene (around 19kb) and do not show any significant flanking sequence to explain its association with obesity (see attach). Thus this SNP seems to be more a marker for the disease than the the real causing variant. nothing for the moment, but still to be elucidated.
fred
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Hi,
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!
Thanks,
Yongyao
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Hello, Yongyao Wang
Morphology preservation depends on your freezing method.
If you use the cryogenic method of immobilization,
such as high pressure freezing,
sample should be preserved perfectly.
Otherwise,
liquid nitrogen plunging may cause severely ice crystal to damage your organelle in the ultrastructure of cells.
But you never know what will happen in EM world,
just do it, you’ll get more than you loss.
FYI
Wolf
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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.
Thanks!
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Hello, here is a paper which my colleague found very useful and it has a nice protocol & shows the gating strategy
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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?
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You should not need to use Matrigel when injecting into the mammary fat pad - as others have said, 4T1 will grow almost anywhere without the additional ECM support. Depending on your experiment, matrigel can potentially confound your analysis. It can be difficult to determine that you have implanted into the mammary fat pad rather than sub-cutaneously near the fat pad, since the cells grow equally well in either site.
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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?
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I suggest that the hypolipidemic effect of gerciana extract be conducted using a model as well as LD50 before trial on human.
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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!
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I had similar observations many years ago in C3H mice. There, our pathologists told me that the ascites was a consequence of injection-associated lipoma. We then moved to tumour induction subcutaneously in the legs of the mice.
W.
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hello,
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?
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Thank you for your help Dongdong Xiao
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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!
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How do you convert NOAEL in ppm to mg / kg / day?
Would anyone have any reference?
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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 .
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Are you evaluating how many viable cells are there before plating the cells? Also, you maybe should seed at a high concentrations, since many cells will die or are already dead, thus, it might be a good idea to concentrate their proportion.
Even so, If the number of cells after the extraction is too low, It might be a good idea to check that you are not digesting the tissue in collagenase for too long or that you are doing the protocol at a good pace.
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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.
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Please check
1. Badaut J, Hirt L, Granziera C, Bogousslavsky J, Magistretti PJ, Regli L.Astrocyte-specific expression of aquaporin-9 in mouse brain is increased after transient focal cerebral ischemia. J Cereb Blood Flow Metab. 2001 May;21(5):477-82.
2. Hui Liu; Mei Yang; Guo-ping Qiu, Fei ZhuoI; Wei-hua Yu; Shan-quan Sun; Yun Xiu.Aquaporin 9 in rat brain after severe traumatic brain injury. Arq. Neuro-Psiquiatr. vol.70 no.3 São Paulo Mar. 2012
3. Jerome BadautLuca RegliLuca Regli. Badaut J, Regli LDistribution and possible roles of aquaporin 9 in the brain. Neuroscience.2004 129:971-981
4. Badaut J, Regli L. Distribution and possible roles of aquaporin 9 in the brain. Neuroscience. 2004;129(4):971-81.
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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! :)
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Yeah! I agree with Ramos, 1: 10 is the standard ratio for tissue and Trizol in RNA extraction. With my experience, Fat or triglycerides will inhibit the quality of cDNA!
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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?
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Why do you not want to use the radiological method? It is very sensitive and is well characterized. I think that if I were going to measure adenylate cyclase activity without P-32, I would try this method. It uses inhibition of luciferase as ATP is depleted.
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Hi,
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!!!
Francesco
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Hi, what's the method you used in the last?
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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.
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When the dimples of Venus are disappearing ( the Michaelis rhomb , used as marker by obstetrics ) where in modern times pretty womans are showing the sexy antlers tattoo, we have an indication for tissue swelling in this aea. This symptom often is connected with fibromyalgia in the lower quadrants, and the kidney meridians are irritated. This is caused by an entrapment of the nerves behind the inner ankles ( ! ). Then the diagnostics for Fibromyalgiasyndrom have to be started, as described in two papers from the Temple University, Philadelphia. After decompression and neurolysis of the entrapped little peripheral nerves the symptoms are disappearing and the fibro also. ( My surgical experience since 1998 - 3 patients the week ). Peers aren‘t allowing publications of method and results. Isn‘t „scientific“. Painfree patients are of no interest. It‘s strange, but the typical indicator for a very effective New Method......
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Hello,
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!
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following
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Hi All,
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.
Cheers,
Jakub
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Hi Jakub,
have you finally found the reason for the non-specific staining and a solution to it? We have kind of a similar problem. We are performing IF staining of bone marrow derived cells using a range of different antibodies. For the majority of antibodies we get non-specific staining of almost all cells. However if we use in vitro samples, we get a specific signal. And also if we use peripheral blood- instead of bone marrow- derived cells, we don`t get this background pattern of non-specific staining. So this problem must be related to the composition of the bone marrow- high fat content???
We have tried blocking, different fixation methods, permeabilization, different antibody clones etc. but this does not solve the problem.
Have you found a way to reduce the non-specific signals? Was it really due to autofluorescence?
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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.
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You can solve to use the nitrogen tank. Place the material in the holder and immerse it in a nitrogen tank with an apparatus for 3 seconds and pull the tissue at an incredibly fast speed and immediately cut the cryostat. We were able to obtain 2 micron thickness sections.
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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?
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thanks for bringing this to my attention. much appreciated.
cheers
John
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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?
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Hi, it looks like you stained extracellular space.
First I would use DAPI staining to find out if the signals are not infiltrated immune cells. Without DAPI staining and information about what is stained with green is hard to tell.
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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?
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Hi Mickey
poor amount of RNA you have. The best approach is the bioanalyzer, but even though you won't have enough RNA.
I can advice you to test one PCR with little amount of RNA, taking a very fresh extracted RNA and one among the RNAs you want to save.
easy, fast and low consuming in your case.
fred
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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.
[1] 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.
[2]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!
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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
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Sometimes those ratios depend a lot on the elution solution which you used. A good old style is just to run a gel and look for smear. Do you have a bioanalyzer in your institute? Its even easier...., if they are above RIN>6 its fine for a qPCR.
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We need to cryopreserve small pieces of human adipose tissue (~250mg) in order to cut cryosections for confocal microscopy.
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We had good success, when we wrapped a small piece of adipose tissue in aluminium foil and then immersed it in N2 cooled isopentane.
Best regards, Daniela
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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?
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Dealing with poor quality RNA is never ideal. If it is possible to re-run the experiments (with larger N so that your power doesn't go super low if a few samples fail), that is always the best solution. That being said, sometimes this is not possible.
What type of gene expression analysis are you trying to do? If you are doing qPCR, you can try running it on your poor quality samples and see if your reaction quality is good by looking at melt curves and the amplification of your housekeeping genes. If you want to conduct RNAseq, while poor-quality RNA is far from ideal, there are methods developed to deal with degraded RNA. Illumina's TruSeq RNA Exome reagents, for example, are design