Science topic

Lung - Science topic

Either of the pair of organs occupying the cavity of the thorax that effect the aeration of the blood.
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Hi,
Im trying to isolate intratestinal macrophages from mouse lungs, and grow them in culture for a few days, to collect secretome. Does somebody have a good protocol for it?
I tried face sorting but the cells didn't grow well and died in the following days.
I also tried to separate f4/80 cells with macs columns but didn't get any separation.
Can someone have any suggestions?
Thank you,
Shani
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Well in that case, you should try supplementing with r-MCSF or L929 supernatant in the cultures. Macrophages are kinda dependent on this growth factor. For more information, please see:
Good luck!
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Please give advices or suggestions here,
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In addition, look after own health first.
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I am using DMEM from gibco for the culturing, but cells seems not happy. I have also tried DMEM Glutamax but in all case cells do not survive more than one month. And the growth is very slow. Is anybody here has any experience in these cells? Please do me help to overcome the problem.
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I think, it is important to know how are you preparing the fibroblast.
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I'm looking for a noisy or blurry COVID-19 CT Scan dataset of Lungs. I would be very grateful if someone helps me to find such a dataset.
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Thank you very much for your Question. Movement can cause CT images to be blurry. That movement can occur in a few ways.
For example, breathing/coughing causes more movement of abdominal contents (and lungs of course), as they rise and fall with the diaphragm. This is the most common cause I see for blurry images because for some people it is difficult to hold your breath for as long as the scanner requires. Of course, there is a movement of the body. Lying flat on a CT table isn’t necessarily comfortable, and some people either can’t or won’t hold completely still.
Best regards
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Dear fellows,
I am looking for an expert in lung histology to collaborate on one of my project.
The expected work is to analyze histology images (e.g H&E, Trichrome, IHC etc) I have obtained from lung tissue from different mice models and provide me with an accurate description, quantification, an proper figure panels of whatever phenotype he/she will observe. The researcher will of analyze all the samples blindly.
For this work the chosen collaborator will of course be one of the co-authors of the future publication using this data.
look forward to here from you.
Best,
Yair
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اعطني قليلا من الوقت حتى اجد من يساعدك
شكرا جزيلا
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I found AAV6 can infect lung, but its efficiency is not so good , improvements are still needed. Does anyone have any idea about the better serotypes than AAV9 for mouse lung infection, maybe capsid mutation or peptide display can screen out some candidate.
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As far as I know, AAV4 & 6 are the most efficient natural serotypes regarding the transduction of airway epithelial cells and alveolar cells. They can be applied intranasally. Efficient application, however, requires some experience. We've tried both (AAV4&6) and only achieved moderate results. With regard to pulmonary (microvascular) endothelial cells, there are AAV capsid mutants that work quite well upon intravenous administration. Our AAV2-ESGHGYF is one example (PMID: 27018516).
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Hey,
I am currently investigating acute respiratory distress syndrome (ARDS) in mice via intra-tracheal infection of mouse lungs with E.coli. Thus far I have validated our model using histological approaches. However, I would also like to have some real time monitoring to assess the progression of the infection/ARDS symptoms. I have been doing peripheral blood oxygen measurements. However the SpO2 % measurements can have high variability across my experiment. the pulse oximeter we use takes quite a long time to read a stable measurement, sometimes I can be stuck measuring a single mouse for 20 minutes. Often, I get two stable readings from my mice. For example, control uninfected mice ideally would have 99% SpO2 saturation. however I can also get a clean stable reading at ~85%. For my infected mice, the values vary. Since there is no standard, its quite tricky to determine which measurement is true. For example, an infected mouse, 6 hours post infection, can have a reading of 77% SpO2 and can then immediately read stably, at 50%.
I am aware of ensuring the mouse is accustomed to being restrained and recovers from that stress before measure blood oxygen. In addition, extremely low stable readings (60%<) can suggest a mouse may need to be euthanized, but qualitative assessment of the mice, exhibits they are are fine and very active, so it makes me doubt extremely low but stable readings.
Has anyone had issues with getting accurate SpO2 readings with mice and have any tips for accurate measurement and interpretation?
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Hey,
first of all body weight loss correlates with the degree of disease. Furthermore, you can evaluate the degree of acute lung injury via albumin and protein levels in BALF in addition to histopathology. For the inflammatory response in the lungs suggest cytokine levels and amount of immune cells in BALF. We used these methods in 2 mouse models of ARDS recently:
Best,
Christian
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Today, it is possible to scan the human body very precisely with the help of x-ray CT images. These CT images are based on voxels. If you look closely at CT images of lung lobes, you can see five lobes segments, the right lung is divided into three lobes segments and the left lung is divided into two lobes segments. The MIP method is one of the most used techniques to define a slap thickness and visualize the desired 2D rendering voxels of lung lobes.
The biggest challenge in this case is how to delete or correct an exact location or a false segmentation of lung lobes without affecting the whole lobes segmentation?
is there a mech technique (like Delaunay Triangulation), which can mesh the entire 2D rendering lung lobesand then select and delete or correct the exact location manually without affecting the lobes segmentation?
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Thank you. I have already read this paper, but it does not help, beacause I have already created volume mesh from CT scan using marching cubes. My problem now is to model a segmented Lung lobe with individualized surface meshes like in the following paper:
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I am doing a comparative study of two different viruses. When I infected mice with the wild-type viruses, the mice will show weight loss but eventually recover. But in case of the mutant virus infection, the mice will eventually die. The mutant virus is, in a way, more virulent than the wild-type virus.
When assessed for the different cell type recruitment in the lungs (infection was done intranasally), we find that wild-type virus-infected lungs had higher NK cells compared to the mutant virus-infected lungs, whereas, in the mutant virus-infected lungs we see higher neutrophil accumulation.
But, I am finding it difficult to connect the recruitment of neutrophils to the fatal consequence of the mutant virus-infected mice. What am I missing?
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Hello Sebastien, we did a proteome array on the Day6 post infection from the lung homogenates of control mice along with the wild-type and mutant virus infected mice. And we see a 2-fold increase in IFNg, TIMP-1 and CCL3 in the mutant virus infected lung homogenate. Which is in keeping with the fact that there is inflammatory response in the mutant virus infected lungs?
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I am culturing Human lung fibroblasts (HFL) in DMEM supplemented with 10% heat-inactivated FBS, 1% MEM non-essential amino acids, 1% penicillin-streptomycin, and 1% L-glutamine. However, they have very slow growth (around 10-15 days to reach confluence in T25).
Is this normal? What can I do to improve their growth rate?
Thanks!
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They are also dependent on the cells' seed dose.
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I am working on Medical image segmentation Problems, I would like to know is there any public dataset available for Lung and Liver images
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Those programs (softwares) are used for estimation of lung dose caused by Radon decay products .
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I have
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Increasing the demand of lead recycling industries in so call E- vehicles or eco friendly initiatives. It was observed that the SOP and the work culture is rarely bothered about the exposure to the labour in such recycling units at melting section, lead oxide mill, red oxide area and ingots making sections. So I want to know about the study conducted on the accumulation of lead in the lungs of the front line workers and is chances to defuse lead from lungs ( respiratory system/track) to blood ??
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The workers in secondary lead smelters lead normally suffer from the lead poison. It is body accumulated poison which causes by the time a sever effect on the lungs, kidneys, blood, bones, and brain. I worked for several years in lead smelters and suffer from this poison. The treatment of such poison is to follow the following points:
1. The importance of staying away from sources of pollution until starts to go down, or reduce the exposure time.
2. Make sure to wear personal protective clothing and focus on appropriate respiratory masks.
3. The lead in the lungs and blood need to be extracted by special medicines, The pomegranate drink is a good extracted liquid for the heavy metals like lead.
I would like you to focuses on the first point as soon as possible in order to get raped to reduce lead poison concentration.
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Hey,
I am planning to dissect rats and isolate Lung tissue samples to be stored in RNA stabilizer for later analysis.
- Should I perform trandcardial perfusion by PBS ?
- Is it necessary to perform perfusion if I want good results ?
- Will non uniform perfusion in different rats affect the results ?
- Is the same crucial for protein expression analysis through WB/ELISA too ?
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Full disclosure; I work on brain, so I'm not familiar with your specific sample type.
That said: The most important thing for integrity of RNA in tissue is to work as fast as possible and as cold as possible. The perfusion procedure usually has a lag of a few minutes between cessation of effective circulation (when the animal is profoundly anaesthetised and heart rate and respiration are very abnormal) and effective cooling of the tissue (once cardiac access is established and cold PBS is running.) During this time, the tissue you want to work on is undergoing hypoxia and oxidative stress, which can make endogenous RNases more active, and could potentially also result in transcriptional changes. For this reason, we never perfuse before collecting tissue when the aim of the experiment is to analyse gene expression - rapid dissection on ice is technically easier, faster, and likely better.
The exception to this is if the presence of blood in your tissue is going to interfere with your results. If it is important for your specific experiment that none of the RNA or protein you're analysing came from blood cells, you should perfuse. I prefer to accept the presence of blood as the natural state of the tissue, because in my opinion there IS a difference in the effectiveness of perfusion between different animals - possibly because I am not very good at this technique, but I guess there is some variation even if the perfusions are done by someone very proficient. I feel like it is more reasonable to assume that tissue dissected without perfusion contains the same amount of blood between biological replicates, versus tissue that was perfused where it might be quite different.
I'm much less experienced with protein work than RNA work but I think most proteins are much more stable and unlikely to be degraded by a few extra minutes taken to perfuse the animal - some post-translational modifications are very labile though.
Hope this helps :)
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I would like to isolate lymphocytes from the lung. One of the step requires a density gradient centrifugation step but most protocols make use of pre-made solutions like EasyColl. Has anyone used a percoll gradient instead? What % did you use?
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40/70% Percoll gradient enrich lymphocytes from lung filtrate
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I would like to know the volume of lining fluid in mice
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Thank you so much!!!!
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We are successful in isolating endothelial cells from Rat lungs and heart. However, due to active endothelial-mesenchymal transition sub-culturing the isolated cells is challenging.
LONZA in their website guarantees sub-culturing human cardiac endothelial cells until P10. Is this true..? Does someone have any experience in culturing them?
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Can I please check if you were able to successfully passage these cells for 10 passages with no mesenchymal contamination?
Thank you!
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Is there any danger of sweat, aerosols or bioaerosols in the case of indoor exercise and/or high-intensity exercise in relation to SARS-CoV-2?
Is sweat a danger?
Are bio aerosols a problem indoors with higher-intensity exercise? Would you have to account for ventilation and air flows?
Is there more danger with heavier breathing patterns or more powerfull ones?
Can you somehow try to make sure if a person has an infection when they do not have symptoms?
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Please check out Dr Bromage's blog posting of environmental risks. Although he did not mention indoor gyms in particular, the virus is easier to spread indoors with poor circulation and poor airflow. The more infected individuals in a single area, and the longer the duration one's exposure...the higher the viral load one is exposed to and the more likely one would be infected.
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In a national level exam, a question is asked that which human body organ involved in the purification of blood? 
The options are (a) Lungs (b) Kidneys
Can anybody give an answer to clear confusion between lungs and kidney? According to me, lungs is the best answer. The kidney is for filtration.
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Kidney is the right option because it ultimately flush off the toxicants whereas lung tied up the excess of acid or base disbalances in the form of buffers until it is ultimately removed by kidney which takes time. And besides maintaining the acid base balance, kidney also removes xenobiotics, drugs and unwanted substances from the blood.
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I am working with an IL22-BFP reporter mouse. IL-22 signal could be detected in gut, but not in lung. Since I am interested in lung expression, i wonder if there is a way to amplify the signal of the BFP-Tag.
I already tried to restimulate the cells with IL-23 and blocking the export of cytokines with BrefeldinA. No signal.
First idea would be an anti-BFP FACS antibody, which is conjugated with a fluorochrome in the same wavelength as BFP.
My search for appropriate antibodies in the internet didn't succeed.
Is there any way to solve my problem and amplify the IL22-BFP signal?
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Hi Frank,
I just realized that I use a triple reporter mouse with three different tags on three different genes: GFP, BFP and tagRFP. Therefore, no matter what version of BFP I use, each possible antibody will cross-react...
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most of the publication recommend a 100 cases of simple procedure before proceeding to VATS resection but no specification of this SIMPLE type .
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I think it means videothoracoscopic pleural biopsy, wedge resections, bulla excisions, ETS etc. Main point is improve ability to using VATS-specific instruments.
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An obvious answer would be tar, but I can't find any analytical evidence for this. I have even found some weak evidence* against this. It seems that it's not even one particular substance.
Haemosiderin is sometimes implicated, but acculumation of this compound is seen equally in smokers and non-smokers.**
*Joyce K. Newman PhD , A. E. Vatter PhD & O. K. Reiss PhD (1967) Chemical and Electron Microscopic Studies of the Black Pigment of the Human Lung, Archives of Environmental Health: An International Journal, 15:4, 420-429, DOI: 10.1080/00039896.1967.10664943
** Craig, P. J., Wells, A. U., Doffman, S., Rassl, D., Colby, T. V., Hansell, D. M., ... & Nicholson, A. G. (2004). Desquamative interstitial pneumonia, respiratory bronchiolitis and their relationship to smoking. Histopathology, 45(3), 275-282, DOI: 10.1111/j.1365-2559.2004.01921.x
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Carbon particles in pollution that has been inhaled.
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We have isolated primary human bronchial epithelial cells and frozen them down. After thawing and culturing them, and having identification done, it appears that we have contamination from the Klebsiella genus. Has anyone had this happen? Would this be something from the donor, or cross-contamination in the isolation process?
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Your welcome!
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FEV1 is measured by spirometry and lung impedance are calculated by FOT/IOS methods. Do these methods have any mathematical relation? Can we interpret lung impedance by knowing FEV1?
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@ tarig: Dear Doctor, even if we assume it mathematically as done. would that "relation" be the same for both healthy and damaged lung? we all know, a damaged lung is less compliant, and so high resistances would be scored to get a small mobilized volume. then we are just talking here of "another" way to calculate the pulmonary compliance !!
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I want to induce hypoxic condition in lung.
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Use Monocrotaline
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I want to perform experiments with animal model of mitochondrial dysfunction in lung. I have seen a variety of OXPHOS defect models for other organs (heart, skeletal muscle, liver, brain etc.) but have not been able to find any specific model for lung. I wonder whether these models also feasible to be induced for lung or if there is any strict model generated for lung. It is also possible to induce mitochondrial dysfunction by cigarette smoke, but we prefer to utilize KO model.
Thank you in advance!
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You should use sheep.
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It is utmost importance to understand the microbiome profile associated with genetic disorders. I studied the composition and function of microbiome profile associated with OSCC tissues compared with FEP controls. In disease with well established aietiology involving genetics and epigenetics factors,microbiome may or may not play a causative role. Nevertheless, they can adopt to the tumour microenviroment while modifying it. Furthermore, their metabolic pathways contribute to increase the inflammation in tumour micro environment by changing the composition to dysbiotic state.That means microbiome profile may influence the prognosis of OSCC. Thus, animal experiments are useful to study all ecological theories in any eco system such symbiosis,dysbiosis, cooperate evolution and competitive exclusion. Of course, CF associated will be able to use for therapeutic purposes (microbiomics) in the era of personalized medicine.
I would like to read all updates on this project.
Dear Scientist, I wish you all the best!
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Thank you. This paper is really useful. I also agree with the authors, the species richness and diversity is less in HIV-bronchiectasis group due to the dysbiotic state in severe disease condition may be due to by P. aeruginosa when compared with CF lung group.Usually in disease conditions pathogen dominates over symbiotic microbes causing less species richness and diversity. In my opinion in these situations metagenomics findings corroborate conventional culture findings.
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Should we perfuse the lung (mouse) with PBS (through right ventricle) to detect cytokines protein by using Western blot?
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Hi Shen-An,
yes, i would need to homogenize the whole lung for Western blot (or maybe ELISA). I'm worried about systemic perfusion will affect the result.
thanks for your suggestion.
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Normal epithelial cell lines
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Thank you all.
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I would extract lung from mouse.
In several tries, i did extract mouse lung.
But i did extract lung very poorly.
So i want to extract lung fast and clearly.
Would somebody give me some tips?
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Hello Minseok,
Here is the PDF attached which can help you with procedure. Method of harvesting mainly depends on the use of the sample. Anyway one tip which worked for me: you should collect blood (at max.) from heart or bleed the mouse first before opening the cavity. It will prevent the things to go blurry or messy.
I hope it ll help.
Best of luck
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Hi
I am studying the role of my protein in lung cancer and using A549 (epithelial lung adenocarcinoma) and H1299 (Non small lung cancer ).
Could someone please suggest what would be the most suitable normal lung cell line against A549 & H1299. Few papers mention using MRC5 (Lung fibroblast) and Beas2B (epithelial bronchus) cell lines.
I would really appreciate your thoughts.
Many Thanks & Regards
Apoorva
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Interesting question - there are really no "normal" cell lines to compare with these cell types (A549 H1299), as all immortal cell lines mimic aspects of cancerous cells e.g. uncontrolled growth, altered apoptosis. I would in any case be wary of comparing different cell types (e.g. epithelial origin vs fibroblast origin). I would suggest using a primary cell type (i.e. not an immortal cell line), such as human lung epithelial cells, and keep below about 5 passages for safety. These can be obtained commercially or cultured from clinical biopsies if the facilities are present clinically.
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I am looking for normal lung cell lines, and will pay shipment costs if someone is able to send them.
The aim is to use normal lung as control for small cell lung cancer. Does anyone know about better controls for SCLC? According to literature the SCLC cells origin is not known for sure (maybe not epithelial cell origin)
Thanks for your help!
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Maybe L132 and MRC-5....
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I did a post mortem on a male mature large white pig that died lat night. Clinically the pigs were non febrile but were depressed, anorexic with a roughened haircoat. 11 other pigs have shown the same signs and died in a similar manner
Post mortem the skin was purplish in color. The both lungs were wet and heavy and upon cutting blood was oozing from the lungs. The cranio-ventral lobe was consolidated/hepatised and fibrin/mucus tags could be pulled out of brochi. The lymph nodes around the intestines were hemolysed. can I please have some dds and possible mangement plan
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Dear
If it occurred accidentally ,you must be thought that poisoning
I hope you do well
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I'm having an hard time trying to evaluate diagnostic concordance between lung biopsy histology reports and radiological findings (CT scan or XRay).. Do you know of anyone that has performed this comparison in children?
If not, how would you proceed?
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i designed an adaptive filter to separate Heart sounds from LS and I need to calculate the efficiency of method measuring SNR , before and after applying the filter.
I want to know the correct equations for SNR in such case.
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Hi,
We have a stock of Murine Leukemia Virus (MLV) but I need to make more. I can't find any good article explaining clearly what MOI they used and what were the culture conditions.
Any suggestions/expert?
My plan was to infect the cells at an MOI of 0.5 and harvest the media at day 4, 6 and 8. Then quantify by TCID50.
Have a good day!
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I think, 0.5 McFarland is enough.
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I am trying to induce l by intraline . So far I have little or no success, most of the mice develop tumor in trachea. Not all the mice develop tumor at same time. Although, I got some tumor in lung , the size and distribution of tumor varies greatly across mice . Can anyone suggests me a protocol ?
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We have succeeded in establishing lung tumors by injecting cells via tail vein.
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Hi
I am working with formalin fixed mouse lungs.
After fixation in NBF (48h; first intratracheal instillation, then in HistoPot) I am dividing lungs into lobes, and then from each lobe I take saggitally cut 5mm-thick slice for further processing into paraffin block.
These lungs, despite fixation, are very delicate, and during these slicing, even when using fresh scalpel blade, I introduce small but evident crush artifacts.
Do you have any hints/tips/advices/ideas how to handle such delicate tissues and not introduce crush artifacts?
(one I have heard of is to first wrap lobe in wet paper towel and cut through it, but in my hands it is still produce some unwanted artifacts)
I would appreciate any advices
Michal
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Hi Michal,
You my try agarose embedding of the lobes after postfixation and before cutting the slice. 5% low melting agarose works well for most of our tissues, but for lungs it might be a bit too dense so check also 4-3% one. Be careful that the poured mixture is not too hot. We usually pour the blocks using agarose at around 40 degrees (checked on the inside of your forearm- just like baby milk :) ). The agarose can be dehydrated and embedded in paraffin with the tissue inside and does not affect sectioning.
Also you my try to cut the samples not with scalpel blade but with fresh, disposable microtome knife. It helps for some people
Let me know if it helps. Also If you have other questions or want to know any details just PM, I am happy to help.
Good Luck :)
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In the KrasLSL-G12D/+;Pdx1-Cre PDAC mouse model, at which time does metastasis start to form in lung and liver? And at what frequency?
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According to the above articles, while most of the individuals die within the short period, some of them who survive longer than 20 weeks exhibit the distant metastasis to the lungs and liver and peritoneum dissemination by all means.
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I'm looking for a good physiological model of the lungs (and thorax), to do some non invasive monitoring of lung function parameters
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Hi Denise
How about something like the little simulation shown in this YouTube video:
I recall doing something similar in high school.
Best regards and good luck
Ian
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I stained lung tumors both with Ki67 and pH3-antibodies and got more stained cells with the first antibody.
I have read several articles claiming different things about which cells are stained with these markers.
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Ki67 is a good biomarker for all stages in the cell cycle, but it is absent in G0 quiescent cells. It could be suggested that Ki-67 does not precisely reflect proliferating cells.
PH3 is more specific biomarker that stains cells in late G2 and mitosis (pH3 stains the condensed chromatin just before chromosomal segregation). For this reason, it is logical to obtain more ki67 stained cells than pH3 stained cells from the same tissue sample.
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I want to learn the techniques for isolation and culture of Alveolar Cells (Type I and II).
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I suggest searchng PubMed ( www.ncbi.nlm.nih.gov/pubmed ) for publications by Dr. R.J. Mason and his colleagues. You will find their publications about isolation of Type II alveolar cells from mouse, rat, and human lungs. For exanple, they published a 1977 publication on "Isolation and properties of type II alveolar cells from rat lung." Disclaimer:This is unrelated to any of my NIH efforts but I did try to isolate Type II alveolar cells during my graduate school research at UCSF.
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I am looking for a research article that has specifically written about histopathological effect of filed cancer in case of major cancer sites such as breast, lung, liver, prostate, colon, etc.
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I am already very deep in literature survey since I am in my 3rd year of PhD. I am currently reading about field cancerization... And since my work focuses on histopathological analysis of images.. I couldn't find any good reference paper with respect to histological changes due to field effect. Hence thought to ask here. Thank you for your suggestion.
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During a badminton step lunge task
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The task is step lunge and i need the point from dominant leg heel contact to toe off. I recently did a pilot, and put an accelerometer sensor on mid shank, at the heel contact point acceleration has a peak but at the toe off point...?
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How to detect collagen deposition in histological preparation of fibrosis sample of lung without using masson trichrome?
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The Sirius Red staining is very frequently used in studies of fibrosis as this technique results as the most sensitive method to reveal collagen fibers.
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I'm having trouble quantifying expression of alpha - smooth muscle actin in human lung fibroblasts.
Culture method: Fibroblasts were cultured on untreated glass slides overnight before addition of TGFB1 (concentrations varying from 0 ng per ml to 10 ng per ml), and further cultured for 72 hours.
I, then proceed to image the samples and I notice a basal expression of alpha - SMA in the 0 ng per ml condition. Upon quantifying the mean fluoresence intensity (MFI) per cell on all conditions: I do not have a high MFI in the 0 ng per ml condition as compared to 10 ng per ml.
Any suggestions to correct the problem? or alternative means of quantifying alpha - SMA through microscopy?
I have tried serum free culture, and I still see a basal expression in the 0 ng per ml.
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Lung fibroblasts normally have high basal level of a-SMA. Most of cells in lung fbl culture are miofibroblasts with good stress fibres (a-SMA) positive, they produce TGF-beta. Not sure you can measure the change of the level of a-SMA by IF in your case. You can probably count % of SMA-positive cells and use WB.
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There was several target agents (Erlotinib (Tarceva) Afatinib (Gilotrif) Gefitinib (Iressa)) for EGFR mutation (+) lung adenocarcinoma.
What agent is the most effective and preferable agent in your experience?
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Dear Dr. Ho Cheol Kim,
this depends highly on the individual genetic makeup of the patient (exon 19 mutations/deletions, exon 21 resistance mutations after first-line treatment, etc.). Third-generation agents such as osimertinib (i.e. Tagrisso) or rociletinib (i.e. CO-1686) represent promising drug/IND respectively to overcome T790M-mediated resistance in NSCLC as two examples. We have just recently summarized the current consesus in terms of targeted therapies against the ErbB family (with a focus on oncogene addiction, which brings targeted therapeutics, as you were asking, into play); see 'ErbB Family Signalling: A Paradigm for Oncogene
Addiction and Personalized Oncology', published in Cancers in April 2017. Hope this helps.
All the best,
Nico
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We have tried the armenian hamster anti mouse and a secondary for rhodamine to use for fluorescence but it didn't work. We wanted fluorescence so we could double stain with another antibody .
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Well, couple of suggestions:
1. Try western blotting, it should work and give you at least some information.
2. Many antibodies (despite claims by the companies) do not work in immunohistochemistry. Many companies such as Santa Cruz claim to have antibodies for everything. My personal experience is that only 50% of their antibodies work. There can be many reasons why the antibodies do not work in immuno.
a) Epitope not available: meaning the site that the antibody bind to is occupied by something else such as another binding protein. Then, you can try antigen retrieval but my opinion is that it is not worth it.
b) different species
c) different tissue meaning different isoforms of proteins etc. Finding one good antibody that really really works in immuno can be a wonderful thing! d) the company is cheating, if some of their antibodies never works you can never figure it out. This is really bad, but you have to remember companies are their to make money. One time I got into a dispute over one particular antibody. Called the company so many times, every time they were saying to change my protocols such as fixative, fixation time, pH etc. After about 20 calls I gave up!
3. You can try other antibodies from some reputable companies such as Sigma.
4. Try polyclonal antibodies.
5. Try different extraction with detergents such as 1% Triton X.
Hope this helps.
Regards
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Dear sir/madam,
I am doing project based on lung sound classification and priorly we have done some experiments on automatic sleep stage analysis. We want to test the same for lung sound classification. But we have identified only paid database by making survey. Kindly suggest some standard database for analysing lung sound and we are eagerly waiting for your response.
Thankyou in advance
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Dear Mahmudova mam,
Thankyou for your kind response .We have made survey and found RALE database for lung sound classification but it is paid . Please suggest some standard lung sound database for free that will be helpful for our study.
Thankyou in advance.
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There seems to be a notion among some oncologists that chemo/immuno therapy for late Stage I lung cancer has less than 6% prospects of effect. While clinical straregies seem absurd to me when based on clinical statistics-- so crude!-- heterogenicity of tumor cells at that stage seems a critical issue. Can anyone expand on this question, as opposed to the crude stats-type oncology? Thank you all in advance for any molecular perspective important to such considerations.
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Hello
We have no clinical evidence of its utility in patients in Latin America. In our hospital we are characterizing the expression of the PD-L1 and we are using it in second line, this due to state regulation.
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I have lung sections stained with Neutrophil ab and DAPI. Thank you
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You can easily measure the tissue area. First, you need to determine pixel to unit (like mm) ratio, for which you need a scale bar on your image. You can set this scale under Analyze>Set scale. Make sure that the "Area" checkbox is ticked in Analyze>Set measurements. When this is set, you can simply click Analyze>Measure and a results window will open with your image area. Or, if you need to measure area of a part of an image, first select your area of interest using an appropriate tool and then click Analyze>Measure to measure your selection. This will help you get the number of cells per area unit.
For more info on above-mentioned steps check ImageJ documentation, it's pretty much straightforward.
Hope this helps!
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I'll be working with normal human lung fibroblasts (Lonza) which will be treated with TGF-b for 24-48hrs. I would like to perform immunofluorescence and qPCR.
Can I please have an idea of seeding densities for 6 and 24 well plates? Also, if it is necessary to coat coverslips with PDL/PLL for IF.
Thanks in advance :)
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In my experience with mouse fibroblasts, seeding 400K cells into a 60mm plate (2x surface area of a 6-well plate), results in 100% confluency in about 48hrs. I usually let them settle overnight to 24hrs before adding any treatment. So if you plan to treat for 48hrs and allow 24hrs for the cells to settle prior to treatment (72hrs total), you may want to start with 100-200K cells in a 6-well plate. However, each cell type will grow at different rates and adding TGF-b may increase the growth rate, so you will have to give it a go and see what happens, then adjust the number of cells when you repeat the experiment. If you are only treating for 24hrs (48hrs total with settling), then starting with 300K cells may be optimal. The idea being you don't want any well to actually reach 100% confluence before you extract RNA, but you also want to have enough cells to get a reasonable amount of RNA for your qPCR, so 85-90% confluence at the time of extraction is the goal.
For the 24 well plates, is that for your IF? Do you plan to put coverslips in the wells and grow the cells on the coverslips? If so, you do want to coat the coverslips with PLL or PDL. The cells may grow on the coverslips without PLL or PDL, but likely not as well and they may detach easily during the washing/fixation steps of your protocol.
For seeding on the coverslips in 24 well plates, I have made a solution of 500--1000 cells per 100uL of media, then I pipette 200uL directly into the center of a dry coverslip (in the 24 well plate) and let them settle for 1-2 hrs in the incubator. Then I add 2mL of media after that and let them grow/settle overnight before starting my assay. This just keeps the cells in the center of the coverslip so they don't grow over the edge and attach to the bottom of the actual well, which can cause issues when trying to remove the coverslip from the well later, in my experience.
On another note, unless the fibroblasts have been imortalized through genetic manipulation or other method, they will only grow well for about 4-5 passages and then will stop proliferating. So timing and planning is important as you will see very different results using fibroblast cells from passage 1 compared to passage 5.
Hope this helps.
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I'm trying to simulate an acute asthma exacerbation with an ASL5000 active servo lung, to model the particle distribution of salbutamol (albuterol) Metered Dose Inhaler vs salbutamol Dry Powder Inhaler. I am hoping to see which performs better at the reduced flow produced by patients at this time. 
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less than 60% predicted value with variability +/-  30%    (for both FEV and PIFR)
absolute volumes depend on body size (age/ht/sex)
.
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I'm sorting for airway epithelium using an mGFP/mTom mouse and an airway specific Cre. When running the WB and staining with Ponceau red, or any other antibody, I get this huge band between 75-50 kDa (around 65kDa) and very few and faint bands throughout the lane. I thought it was albumin, since my sort goes into 2% FBS-PBS but I wash twice with PBS before lysing them. In addition, my other population, the non-airway one, looks and behaves perfectly, and they are both treated exactly the same. After sorting, I count my cells using a haemocytometer, and they look healthy. I load my lanes by number of cells, not by protein concentration. Any help would be greatly appreciated. My protein of interest is 68 kDa. Thanks!
Image: Lane 1 is airway epithelium 750k cells. Lanes 2, 3 and 4 are non-airway lung cells 500k, 750k, 1 million cells respectively. 
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Thank you both. Hopefully will get some answers soon.
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In the acute toxicity trials of some pesticides, an increase in the weight of various organs such as the heart, lungs and liver occurs, but the weight of the spleen decreases. What does this mean?
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Let me guess: inflammation in numerous organs leads to massive recruitment of blood cells into inflamed organs. Bone marrow can't keep up. Blood cells get depleted and the spleen that filters the blood now contains significantly fewer blood cells. As a result the spleen weight decreases.
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I am using NCI H727-a non-small cell carcinoma of the lung cell line and the cells are growing very slow, can someone help troubleshoot here? Is there any way by which we could make the cells grow relatively faster? I was reading articles online but, haven't gotten any much information. Has anyone worked with this before and can guide. Thanks! I am following the methodology described by the ECACC using RPMI 1640 as the medium.
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Not yet.. will do that! According to the literature, I have read, the cells grow relatively slow...thank you for the advise!
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I will use the EnzChek Protease Assay Kit to detect proteases activity in bovine lung tissue.  I have never worked with lungs and I would like to use a sonicator to isolate proteins (do I actually need to do this?).  I am not sure if I should use a lysis buffer with a protease inhibitor or not. Does anyone has experience with proteases detection? Any help will be much appreciated!!
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If you add protease inhibitors to your tissue homogenate, you may inhibit the very protease(s) you are trying to detect. Don't use protease inhibitors unless you are sure that your particular protease is not affected by them.
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Hi, does anyone have an information regarding further treatment options for a 61 year old male with mCRC with secondaries to the left lung and mediastinal nodes who has DPG deficiency confirmed after two doses of Avastin and Folfox when he presented with neutropenic sepsis/typhilitis and local perforation. Any options other than single dose irinotecan?
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Hi, try with mitomycin C plus irinotecan, or with regorafenib or pemetrexed. You can find several phase II trials suporting this therapies. 
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What is the best method to transplant macrophages into mouse lung after original alveolar macrophages are depleted? Do I have to use BMDMs for transplantation or alveolar macrophages can be used? please suggest me with a good reference
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I'm trying to isolate type II alveolar epithelial cells. I've attempted to use a JOVE protocol but found that not only was my cell number low (100,000 cells per mouse lung), my viability was ~20% by trypan staining. I shifted to the another published protocol using MACs products (automacs and Macs Columns) and my cell number increased, however by Trypan Blue and PI - my cells appeared to be alive (~85% viable) but with Annexin/PI staining my cells were truly only between 10-30% viable. My most recent enrichment is using EpCAM PE positive selection (magnets), and again my cell numbers increased- but my viability remained low. Typically, my purity (checked by EpCAM and intracellular SP-A) is okay. However, I'm unable to culture my cells due to the low viability. 
I've listed the steps I'm using below, has anyone had better luck isolating and confirming viability by Annexin/PI with another protocol or one of the protocols above? Is Dispase, liberase, collagenase, or another enzyme better for the initial digestion? Any suggestions are greatly appreciated.
The current protocol is as follows:
1) Sacrifice mice with Avertin/cut renal artery
2) Perfuse with heart with 10 mLs PBS until lung is clear
3) Instill through the trachea 3mLs Dispase (pre-warmed - 5000 caseinolytic units) followed by 0.5mL of 1% LMP agarose
4) Cover lung with ice and let harden for 2-3 minutes
5) Lungs removed with forceps and placed into a 5mL tube with 1 mL pre-warmed dispase for 6 minutes
6) Mince lung by cutting with scissors, incubate at RT 10 minutes in 5mLs UltraCulture media and DNase (200 ug/mL)
*alternatively, I've also tried using a gentlemacs to create a single cell suspension in the same media mixture and found that the cells were also unhappy in that.
7) Cell suspension is filtered through 100 uM and 40 uM filter and rinsed with UltraCulture media and centrifuged 10 minutes at 200xg
Additional selection occurs to enrich the cells, but typically the viability only decreases from ~30% at this point- making me believe that there are problems in the initial digestion. Again, any suggestions would be so helpful!
Thanks!
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Thank you both! 
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How much do kinetic parameters vary across tissues like lungs, liver, brain etc.?
Please add any supporting papers if any.
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Crystalline enzymes should have the same Km across all tissues.
there would be variations depending on the methods of purification of the enzyme.
eg an old Biochemistry text (White Handler  Principles of Biochemistry McGraw Hill 1959)  has a table showing some Km variations between different mammalian tissue for the same enzyme and substrate. see attached
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I will wish to isolate primary AEC from murine lung but I can't find the publication of Corti (1996, Isolation and primary culture of murine alveolar type II cells). Does anyone have this publication or a detailled protocol to prepare mouse alveolar epithelial cells please ?
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Thanks very much for your quick answer.
I come back to you if I need anything else.
Sincerely, Delphine
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Does anybody know a protocol to deplete lung microbiota? If so, do you know if the protocol modifies intestinal microbiota?
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I don't have an answer, but I have the same question. Do you have any clue by now?
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I want to grow lung metastasis from EO771 cells injected into the mammary fat pad. My mice are on C57Bl/6N background and I wonder if anybody knows maybe if EO771 are also from Black6/N or J mice...
It would also be helpful to know if anybody has maybe experience with lung metastasis from EO771. I have trouble getting some. We operate the primary tumor and wait for mice to get sick. However this seems not to happen. So I wonder if it would maybe be better to not operate at all... or alternateively to inject intra venously.
big thx for your help.
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Thx a lot! :)
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Hi everyone! I am trying to perform IHC for F4/80 (ab6640 abcam) on pfa fixed LUNG samples, but i'm not able to obtain a good staining. Do you know a good protocol?
I usually use a concentration of 1:100 and I perform heat-mediated antigen retrieval using citrate buffer, pH 6.0. Thanks in advance!
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For monoclonal clone MCA497GA (presentation of AbD Serotec/BioRad/Biosciences) we use a 1:100 dilution.
Ag retrieval with  20 micrograms/mL Proteinase K (Roche) solution 15 min/37ºC (humidified incubator), diluted 1:1000 in TE, from an stock at  20 mg/mL
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Hi everyone
Would anybody be able to tell me if it is possible to culture cystic fibrosis bronchial epithelial (CFBE) cells at ALI on transwell inserts? I have found plenty of literature that describes the successful culture of CFBE polarised monolayers using liquid-liquid interface (LLI) but nothing to date for ALI. 
I have attempted to grow CFBE monolayers using 3 days under LLI and then switching to ALI. There is a monolayer of cells visible but the TEER is very low, even after 14 days of culture. Has anyone experience with this? Specifically, the optimal culture time under LLI before switching to ALI would be very helpful.
Thanks in advance,
Alan
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Not too late at all Matt, many thanks for your help!
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I am looking for an immortalized non-tranformed lung cell line in which I can induce EMT using TGF-b1. 
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Thank you Sir.
I am looking for a 'kind gift' from some one if possible. 
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Only trying to make connections...
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Thanks. I have read It.
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Does anyone have a value for Rat and/or Human Nasal microsomal protein concentration (mg/g tissue)? No publications actually put the protein concentrations, just that protein concentrations were determined. This would be a HUGE help!
Thanks
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Typically enzyme kinetics is done using microsomes from rat nasal olfactory tissue, or olfactory mucosa and sometimes microsomes from nasal respiratory epithelium. - Most papers publish the kinetic data, but do not report the concentration of microsomes tested (for example, it will just say in the methods that "microsome protein concentration was determined by a standard BSA assay."  --  I need to know the microsomal protein concentration for these tissues. - From what I have seen in the literature, others just use the concentrations of microsomes in the lung for nasal scaling because the values for the nasal tissues are not reported. I just want an idea of whether using the values for lung are in actuality way off, or if that is fine. - There are lots of labs that isolate nasal microsomes, and I am hoping that maybe somewhere in someone's notebook, they have recorded the protein concentration! -- Thanks!
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Lung Imaging
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Yes i agree with Prof Sanad,receptor persent in lung are the best for diagnosis of particular disease by phramaceutical imaging via SPECT and PET..
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I just planned study with rat lungs. I had a good result with formaldehyde fixation. I recently notice paraformaldehyde (in PBS) used for lung fixation by researchers and  they generally arranged fixation time 4 hour. I wonder can lung tissue wait in PBS about 1 week after 4 hour PFA fixation. and then I start tissue processing.   
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Dear Yasin,
as long as AND at least you don't fix the rat lungs initially by state-of-the-art perfusion fixation I guess that "fixation" results after 4 hours( @ 4°C?, @RT?) penetration time (naturally depending a bit also on the specimen's size) are much too short  (for a "perfect fixation" in state-of-the-art and 'chemical terms',  see /cf Kiernan paper on:
John A. Kiernan: "Formaldehyde, formalin, paraformaldehyde and glutaraldehyde:
What they are and what they do." 
Article published in Microscopy Today 00-1 pp. 8-12 (2000). [Copyright (c) 2000 by Today Enterprises. Reproduced with permission.
For histology and/or IHC I would recommend at least [buffered FA/NBF-] fixation for 4-5 h at RT, followed by at least fixation over night [usually then done at 4°C....traditionally (:-))  ], then specs  are removed from the fridge - warming to RT for further 1-2 hrs (best using always specimen rotator) - follow recommendations according to Kiernan! -   up to 2-3 days, in the refrigerator also 1-2 days longer. When correct fixation then has been achieved or can be expected to be finished (always must meet your examination task!, i.e. Histology-Morphology, IHC, ICC, RNA-DNA matters, IF, CLSM....,etc,. etc., 1-3x washing in PHOSPHATE BUFFER solution (pH 7.2 - or 7.3 - or 7.4), also whole washing procedure(s) can last  up to 2 days (rec.@°4C), then you easily and cheerfully can start the tissue processing (guessing into paraffin...). So called "overfixation" is - for sure - not to be expected, except you have to deal with a tricky, delicate Immuno-biomolecular task.
If you intend to use specimens for classical histochemistry or IHC, you could add still in the second buffer washing solution e. g. + 50 mM NH4Cl [Ammoniumchloride) for blocking free aldehyde groups (as well as quenching autoflurorescence -if present and otherwise troubling). Good luck and best wishes, W. M.
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  • For example medical image security needs that reversibility,authentication so on. In my work, I detect the cancer in CT lung and I need to know what the requirements for detection the cancer in the lung(especiale pulmonary nodules) so I hop the answer is supported by reference if you please.
  • Thank you
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There are so many forms of malignant tumors of lung from bronchial, pleural, apical and of many different types of histopathology, cytology, different modes of spread. Only CT is not enough for diagnosis and management. Most important is early diagnosis and surgery. Total cure is possible with early surgery and accurate diagnosis and excision of lymphatic spread by checking frozen biopsy during surgery..
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Hey, I am searching commercially available hydrogels or other substances for 3D culture. Here, it is important that I can individually change the young modulus of the scaffold, but I assume this shouldn´t be a problem. I already found some suitable products, but I am now interested in your experiences.
I would really appreciate your advices. Thanks
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You can use GelMA (methacrylated-gelatine) for this purpose. By playing around with the concentration of the gel and or degree of functionalization (DoF) you can greatly influence the youngs modulus and have cells growing in it.
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A 58 years old male presented with history of right sided chest pain and cough 2 years ago with CT Attached(1). CT guided taken outside our hospital with pathology suggestive of adenocarcinoma lung. Slide review done on our centre rejected this diagnosis and said that there are no malignant cells. Repeated CT and CT guided biopsies 4 times were not diagnostic but only necrotic tissues. Attached here is the last CT.(2-3). During these 2 years duration of hesitations, 2nd and 3rd opinions , he was complicated with right sided empyema and septic shock complicated by acute renal failure that mandated CRRT for 3 months. Now renal functions are normal. He is asking for help and not hesitant as before. What can we do for this patient as we have 3 options; 1st just VATS biopsy and that set. 2 nd is open biopsy only. My impression is thoracotomy exploration, biopsy, excision of this right lung mass through right upper lobectomy, bilobectomy or even pneumonectomy or sleeve resection,
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Hi
Great case.
It all depends on the resources you have. On the second image you show us, it looks like there is an airway adjacent to the mass. I would probably attempt EBUS with TBNA and have rapid on-site pathological evaluation, if available.
If surgery is more readily available than bronchoscopy, I would do PET/CT scan followed by surgery if FGD avidity is high. 
It doesn't look like VATS would be of high yield here. 
Hope it helps!
Keep us posted about the results.
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I want to compare lung cancer cell lines and normal lung cell lines for my research so please can you suggest generally which normal lung cell lines are preferable for research.
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It is highly depending on the type of research you are doing: cancer ? Pulmonary disease ? etc...
Normal cells include fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, etc...
Here attached you have some examples of the type of "normal" lung cells we used in our cancer-related experiments.
Best regards
Robert
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Patients with primary breast cancer can develop distant relapse to the bones, brain, liver and lung. The golden standard to diagnose the lesion and assess its receptor status is a biopsy. However, there are cases in which biopsies cannot be obtained due to the location of the lesion. Do you know how often it is the case that a biopsy cannot be obtained? Thank you!
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To my knowledge, no spesific study on the issue you pointed out, exists.
We can however get some insight from biopsy studies:
Bone biopsies seem to be most prone to failure (36%) and 20% of overall biopsies are considered to be insufficient due to various reasons. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015424/)
Complications are associated in 1% of biopsies (https://www.ncbi.nlm.nih.gov/pubmed/24508104/)
About 1% of patients willing to undergo biopsy do not get a biopsy due to safety issues (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046433/)
Overall the percentage of patients from whom (a high quality) biopsy cannot be obtained may be as high as 20%. Only 1% of cases is considered so risky that a biopsy is not attempted.
As you see, to get a definite answer, you need to focus the question. I hope this was of some help. Maybe an interventional radiologist could give a more practical insight.
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Does anyone has the problem with bioluminescence? The A549 cells were transduced with Firefly luciferase + IRES-mCherry Lentifect™ Purified Lentiviral Particles and inject into the nude rats lung, but no signal after injection with the D-luciferin (150ug/g) even there is a 80mm tumor in the lung.
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Did you check that the cells were bioluminescent after lentivirus vector infection, by imaging them in a dish even before injection?
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I am looking for How lungs have got an association with liver..what is the underlying mechanism that causes Intrapulmonary dilatations from portal hypertension????
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It is not just dilation. there is also aberrant vasculogenesis in HPS; probably VEGF driven. The basic connection seems to be an overflow of the factors driving splanchnic vasculogenesis to reduce portal hypertension, into the pulmonary system leading to undesired aberrant vessel formation and dilation of existing vessels. This is seen in experimental models.
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