Questions related to Lung
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?
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.
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.
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.
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?
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?
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?
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?
I am working on Medical image segmentation Problems, I would like to know is there any public dataset available for Lung and Liver images
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 ??
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 ?
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?
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?
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?
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.
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?
most of the publication recommend a 100 cases of simple procedure before proceeding to VATS resection but no specification of this SIMPLE type .
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
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?
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?
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!
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!
Should we perfuse the lung (mouse) with PBS (through right ventricle) to detect cytokines protein by using Western blot?
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
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!
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
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?
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.
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.
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!
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 ?
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
I'm looking for a good physiological model of the lungs (and thorax), to do some non invasive monitoring of lung function parameters
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.
I want to learn the techniques for isolation and culture of Alveolar Cells (Type I and II).
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.
How to detect collagen deposition in histological preparation of fibrosis sample of lung without using masson trichrome?
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.
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?
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 .
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
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.
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 :)
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.
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.
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?
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.
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!!
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?
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
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!
How much do kinetic parameters vary across tissues like lungs, liver, brain etc.?
Please add any supporting papers if any.
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 ?
Does anybody know a protocol to deplete lung microbiota? If so, do you know if the protocol modifies intestinal microbiota?
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.
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!
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,
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!
lung funcion in non smokers with copd like disease
lung funcion afther specific tratment
identifiing ethiopathogenic factor
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.
- 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
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
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,
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.
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!
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.
I am looking for How lungs have got an association with liver..what is the underlying mechanism that causes Intrapulmonary dilatations from portal hypertension????