Animal Cell Culture - Science method

Thank you so much Lina, i will give it a try Lina M. Yanygina

hai Malcolm Nobre thank you very much for your answer, this answer so helpful for me~

i hope you always stay health

Hi, i know its been a while but, how did it worked for you?

Hello Alif Ismail

In order to avoid toxic effects of DMSO on cells, DMSO concentration should be ≤ 0.1% when it is finally added to the culture.

Suppose your working concentration that is finally added to the culture is 10uM, then you will have to prepare a stock of 10mM of your peptide dissolved in 100% DMSO. When you dilute your stock (10mM) 1000 times with culture media, you will obtain 10uM of working solution. At the same time, 100% DMSO will also get diluted 1000 times giving a concentration of 0.1%. So, you will have 10uM of peptide working solution in 0.1% DMSO when you finally add it to the cells.

If you have limited peptide sample, then you will have to run a small experiment to determine the level at which DMSO toxicity begins. Some cell lines can tolerate up to 1% DMSO without severe cytotoxicity. If MDA-MB-231 and MCF-7 cell lines can tolerate up to 1% DMSO, you may be able to save peptide sample for another assay because you will now be able to make a stock peptide solution of less than 10mM.

Best.

Hai Ping Yu

i have some trouble with my neutral red assay. I get highly variable values in my cell free controls (only medium) and i think it has to do with precipitation of the dye. Did you have any luck with your trouble shooting? Help would be highly appreciated :)

Isolating lymphocytes from a mouse spleen involves several steps:

1. Spleen collection: Harvest the spleen from a mouse and place it in a sterile container with cold PBS (phosphate-buffered saline).

2. Spleen dissociation: Mechanically dissociate the spleen into a single-cell suspension using a tissue grinder or a syringe.

3. Red blood cell lysis: Remove red blood cells by adding a lysis buffer (e.g., ACK lysis buffer) and incubating for 5-10 minutes.

4. Centrifugation: Centrifuge the cell suspension at 300-400 x g for 5-10 minutes to pellet the cells.

5. Cell washing: Wash the cells with cold PBS to remove any remaining debris.

6. Density gradient centrifugation: Layer the cell suspension over a density gradient medium (e.g., Ficoll-Paque) and centrifuge at 400-600 x g for 20-30 minutes.

If your cells are in PBS they frequently do not pellet; add BSA or serum (1%)

Since the enzyme is secreted, it's the extracellular enzyme that matters, so you don't have to worry about how well the inhibitor penetrates into the cells. Besides the IC50, you also have to worry about the inhibitor becoming bound to medium components such as albumin or lung surfactant, if those are present. This binding will reduce the potency of the inhibitor, because only free inhibitor is capable of inhibiting the enzyme.

The treatment dose should be determined experimentally by varying the inhibitor concentration and measuring the effect at each concentration. You can then relate the effect in the cell culture to the IC50 for enzyme inhibition, if you wish.

There are a few strategies you can try to improve the filtration process:

Pre-filtration or Centrifugation:

As you suggested, centrifugation can help remove larger particles before filtration. You might need to optimize the centrifugation conditions, such as increasing the speed or duration.

Pre-filtration through a larger pore size filter (e.g., 80µm) or a cell strainer can also help remove larger particles before using the 0.22µm filter.

Multiple Filtration Steps:

Consider using a stepwise filtration approach. For example, you can first filter through a larger pore size (e.g., 0.45µm) and then use the filtrate for a subsequent filtration through a 0.22µm filter. This can reduce the load on the finer filter.

Filter Washing:

If the filter becomes clogged, you might try washing it with an appropriate solvent to remove the accumulated material. This could be done during or after the filtration process.

Use of Filter Aids:

Some researchers use filter aids to enhance filtration. For example, diatomaceous earth or filter aid pads can sometimes be used in conjunction with the filter to improve flow.

Optimizing Chelex Resin Chelation:

Ensure that the chelex resin chelation process is optimized to minimize the presence of particles. You might want to try different incubation times or concentrations to find the most effective conditions.

Thank you again for your valuable insights Sabbir Khan

V H Krishna Prasad No, calcium ions can affect cell adhesion.

The simple answer is NO! Even if they have been frozen with DMSO they will change (even morphologically). I know the very hard way !!! In my case, adherent cells began to grow only in suspension and lost adherence property. Also, you can have significant cell death and recover only a fraction and it will in essence be a "selection" from within the population.

If they are irreplaceable cells, and critical, you can grow them. BUT to use them you must (1). Have not much loss in viability so you can recover a good representation of the population. Otherwise you are selecting for survivors.(2). Have previous assays that you can confirm that the cell behaves phenotypically as expected. Like a drug response, response to growth factor etc. This is critical if you want to reproduce previous results you got with the cell line.

Even otherwise, if they are critical cells and recoverable, you should note all the steps you perform to recover them, and report that accordingly.

Good luck!

Dear Dr. Dieni

You may use a plate shaker with a slow speed of about 25-50rpm.

Best.

Hi Shaktiprasad, I am the Product Manager of the neural team at STEMCELL Technologies. We need a little more information and context for your question. Please contact our Product and Scientific Support team at techsupport@stemcell.com and a team member will troubleshoot with you.

Kind regards, Nusrat

The CO2 regulator of the seldom - used incubator may be out of calibration. Too much CO2 would turn the medium acid/yellow and affect the cytoskeleton/morphology. One example:

Here is a nice review:

You could possibly also allow your cells to attach for 30 min at RT, leaving the plate in the biosafety hood before placing the plate into the incubator.

@ Vikram Kumar,

Of course, it depends on your protein of interest, but some proteins are mainly in complexes with other proteins. If such a protein is over-expressed alone, it is not very stable and can aggregate.

We had examples in our lab where mammalian proteins, upon overexpression in E. coli, always had very specific "contamination" with a bacterial protein. Using mass spectrometry, we could find out that the overexpressed protein formed a complex with a bacterial homologue of a highly conserved protein, which turned out to be a binding partner of our protein. The fraction of overexpressed protein, which was not bound, simply was not soluble and could not be purified.

So if you know that your protein is "not happy" (i.e. very unstable) without a certain interaction partner, it makes sense to co-express that interaction partner.

Hepatocytes in the liver express and secrete a great deal of protein, including serum albumin, which is the most abundant protein in blood plasma.

Do not centrifuge. Add complete media and then after a day when the cells are adhered wash with 1XPBS and add new media. In this way you may minimise the cells which are already under a lot of stress due to freezing/thawing.

Hi Evelina Lučinskaitė,

I grow primary lung epithelial cells and use TryplE to lift the cells.

I don't really have a problem with the "long" incubation time (I use 5 minutes), however sometimes when my cells are particularly well adhered I need three incubations (this doesn't seem to affect viability particularly).

In saying that, I've not used accutase much, so I can' really comment on the comparison of the two.

Best of luck!

Sam

Dear Talita Rocha ,

regarding your question from a few years ago, I would like to know if you have solved your problem with HaCaT. And how? Thank you :)

Hi Ravichandran Subramaniam and Jessica Dal Col could you able to identify what were these black dots? I my lab, we have been encountered this problem in a549 cells, so I wonder whether they are stress related or any chance are these contamination. Thank you so much for your help :)

Hi Deepdarshan.

BEAS-2B is a human lung epithelial cell line that shows prominent inflammatory responses after induction with Dust. As I am working on Fine particulate matter induced lung inflammation So I would suggest you to plate 8-10k cells per/well in a 96 well plate and check for the cell viability with varying doses of dust particles treatment. Further you can choose the most significant dose where cell viability is reduced and there is an increased expression of inflammatory proteins and an decreased expression of anti-oxidant proteins (can be done by immunoblotting of immunofluorescence). You can also measure the ROS by CM-H2DCFDA or MitoSOX to conclude the oxidative effect of dust particles in lung epithelial cells.

Thanks

Hope it helps...!!

Someone else said low pH but also high pH if it's a yeast contaminant. So observing the color of your media is key.

Things only get cloudy if the contaminant is far along.

Hello Vikram,

You should definitely not use it because there is a possibility of reproduction of some microbes. In this way, you increase the risk of mycoplasma infection.

Best

Yes sir I'm maintaining both the cells in ExpiCHO media and 8% CO2.

The cell will drift over passages. Usually, I would use the cells within a 5 passage range for one study. Doing cell authentication before and after the study is recommended and sometimes required. I rarely use cells above P20 for any cell line. The longer your passage an immortal cell line, you are selecting for the faster growing population of cells. Prepare a low passage master cell bank and a large working cell bank for each cell line. For each experiment, thaw a vial from the working cell bank and replace cells after 5 passages from thawing. This way, you will have a more consistent result.

25 years ago, it used to work. We were feeding our quartz distillation machine with bulk RO water (in-house supply) which had been prepared from Munich's municipal water. Media then were filter sterilized into autoclaved bottles. No UV treatment was involved.

Depending on the price for off the shelf liquid media, you might want to calculate the efficiency and cost of the whole process, though

hello. In most cases, it's normal. when you store cells immediately in -80 without using foster 40, there are chances that FBS which is in freezing media gets precipitated fast. nothing to worry about. once you thaw, it will be fine and cells will grow. It is not the contamination.

Hello Vikram Kumar

Besides your examples, I would like to add a few more.

Succinylation, malonylation, Sumoylation, S-nitrosylation, Amidation, Hydroxylation, Palmitoylation, Glutarylation, Crotonylation, Sulfation, Formylation, Myristoylation, Glutathionylation.

Please refer to the article below for more information.

Best.

Kavitha Jade Brunner it sounds to me like you're doing the right things regarding the water in the water baths. As for cleaning surfaces, using the bleach wouldn't cause contamination, it can just wear down on the surfaces over time.

Disinfecting the scope and surrounding areas as best you can will likely help quite a bit! I'd love to hear if it helps as I am quite curious as to your contamination source as well, now.

I'm not sure why you don't filter media, but perhaps there is something about these cells/this media that I'm unfamiliar with, which is certainly a possibility! Since you're using antibiotics, it can be good to regularly test for mycoplasma as well if you don't already. Though you may not have visible contamination in all your flasks, contamination can sometimes be masked by antibiotics. It seems to be a point of contention among scientists (much like "a-POP-tosis" versus "a-PUH-tosis" haha) but I personally prefer to culture cells without antibiotics. If your aseptic technique is good (sounds like yours is - your source of contamination is likely from the dissection conditions), there's really no need for antibiotics in my opinion! Your PI may vehemently disagree, but that's just my two-cents. Good luck and feel free to keep me updated as to whether the scope disinfection helps!

Hello

l don't know how researchers get splenectomy from the mice where is alive

May be doing surgery but l am getting splenocyte from mice after euthanized means removal of spleen in biological safety cabinet to make splenectomy in tissue culture raw

As you suggested, pH would be my first thought. For glutamine, there will be significant degradation in 24hr at 37°C which would be accelerated in the presence of 10% FBS, but I don't think the effect would be that drastic. Anyway, the cells are cultured in the same medium at 37°C for days. You can run an experiment with control medium warmed at 37°C for 24hr outside of the incubator (without CO2) and compare with the medium pre-incubated inside the incubator (assuming it is not in an air-tight vessel/container).

Also these are good:

1- Writing Research Papers. Student's Book: From Essay to Research Paper

Dorothy E. Zemach, Daniel Broudy, Chris Valvona - 2013 - 128 pages

2- English for Writing Research Papers (English for Academic Research)

Part of: English for Academic Research (11 Books) | by Adrian Wallwork | Mar 3, 2016

3- https://www.scribbr.com/category/research-paper/

One reason cells clump is because DNA molecules are released when cells die in culture, and because DNA is large and negatively charged, cells stick to it and form clumps. DNAse I has been used to declump cells. I have used it with some good and some not so good results. One thing that works really well is a Miltenyi product, their Tyto Running Buffer solution really does prevent clumps - so much so that I use it when I thaw cord blood MNC preps that have been stored at -80oC for over 3 years and had very good recoveries. It's not cheap - over $1USD/mL, but well worth it.

DEar Vikram Kumar

As Jitender Jitender told, the Expi cell lines (both CHO and 293) are cells respectivelly derived from the CHO-S and HEK293.

Since those cells are proprietary of Thermo, there are not many information about the modifications. It seems that the cells were not subjected to any genetic modification but just adapted to growth in suspension in the rich and BSA free EXpi expression mediums which allow to them to growth until very high cell densities and therefore reach high specific protein expression yields.

In my experience both EXpi cell lines are certainly expensives but very powerful.

In my experience, the Expi293 work very well for the production of recombinant proteins or fab antibodies while the EXpi-cho guarantee higher yields for expression of full lenght mabs.

You can find some more information about if on my blog (PROTEOCOOL):

Ciao

Manuele

Maybe the link can help.

This is experiment no or the clone no.

Fromm your experimental setup it seems you are inducing toxicity in various cell lines and trying to analyze cell viability, ROS levels etc. I would suggest you to analyze Nitric oxide (NO) level along with ROS to confirm whether your protein is inducing stress-mediated cell toxicity. Once it is confirmed that ROS and/or NO are elevated, then you can check antioxidant enzymes like SOD, Catalase etc to correlate the cellular antioxidant enzymes with ROS as well as NO level that may signal downstream pathways to initiate cell toxicity including apoptosis, autophagy, necroptosis ans well as necrosis depending upon the extent of oxidative stress insult.

Once you finish the above parameters then you can check the pro-apoptotic and apoptotic markers such as p53, Bax/BCl2, upstream as well as downstream caspases (Caspase 9, Caspase-3 etc) and DNA damage by agarose gel electrophoresis. Its always better to have morphological changes at the level of cell in vitro (I mean morphological apoptotic features such as blabbing, shrinkage, cytoplamsmic fragmentation etc) that can be very useful to correlate the .biochemical well as molecular data as described above.

Although there are several other important parameters that could be analyzed but you have to see that whether your lab facilities/resources allow you to conduct proteomic and genomic level analysis. Good luck

FBS should be stored at -20C for long term storage, but making complete media and storing that in the fridge is not a problem. This is how routine cell culture is normally done. I make a whole bottle (500 ml) at once and store that in the fridge with the FBS and any other components mixed in.

Hi @Vikram,

I proposed such solution for the reactions that not require a special atmosphere (ELISA, CFR...). You are right, it is inapplicable in your case, but you can increase the relative humidity by putting a container filled with water at the bottom of the incubator.

Manuele Martinelli , Wolfgang Schechinger thank you

Fcs alternatives for HL 60 neutrophil like cell line is FPS (slightly different).

study.com/academy/lesson/adherent-suspension-cell-cultures.html#:~:text=Adherent%20cells%20grow%20by%20remaining,of%20a%20tissue%20culture%20flask.&text=Suspension%20cells%20will%20float%20and,be%20mechanically%20or%20chemically%20removed.

Dear Vikram Kumar,

You will receive a product sheet with all of the cell lines you have purchased, which contains detailed information on how to handle the cells. The product sheet also includes batch-specific information like the number of cells per vial, the recommended split or subcultivation ratio, and, if available, the passage number. Prepare the appropriate medium, serum, and other reagents for reviving cell lines by assembling the necessary growth medium, serum, and other reagents. Many of these items are available from suppliers and can be combined with cell lines to create a custom order.

After receiving your desired cell line, whether adherent or suspension, thaw the cell as directed on the product sheet, count the cells using a hemocytometer, and reseeded into a culture flask based on the cell count. Finally, examine the culture flask under an inverted microscope the day after it has been thawed to look for any anomalies.

A new cell line is being studied. Even if it has been done before, it is better to repeat the procedure by performing a simple karyotyping test because there is a chance that some important characteristic of the cell has been lost due to prolonged passaging.

If the cell characterization has been passed, then propagate your desired cell line and prepare for the stock by using the recommended freezing media according to the product sheet of the supplier and transferring the frozen cell line into the liquid nitrogen vapor phase as early as possible.

Hi Ravichandran

Check Merck for effective protocol

Hello Vikram Kumar

I would like to just make a correction in your statement about trypan blue. Trypan blue dye will be excluded from membrane-intact live cells but can enter and concentrate in membrane-compromised dead cells, rendering the dead cells dark blue.

You can use cell-impermeant DNA binding dye, such as propidium iodide. A healthy living cell has an intact cell membrane and will act as a barrier to the dye so it cannot enter the cell. A dead cell has a compromised cell membrane, and it will allow the dye into the cell where it will bind to the DNA and become fluorescent. The dead cells therefore will be positive, and the live cells will be negative.

You can also use the live/dead fixable stains, also known as amine reactive dyes. This also works using the principle of cell membrane integrity, where amines on the outside of a living cell with react with the amine-reactive dye and have a dim fluorescence, while a dead cell with a compromised membrane will allow the dye into the cell where it will react with amines throughout the cell resulting in a bright fluorescence.

Best.

Dear colleaque the question clear,need the alternative of sod.citrate to suspend chicken blood,so the answer the following two alternatives are :

1 _Acid citrare dextroseACD Solution ,allow 1 part of ACD Solution to 3 parts of blood.

2_Alsevars solution,allow 1 part of Alsevar,s solution to 1 part of blood.

Best regards

Thank you Kosheeka Primary Cells

In addition, such an experiment would be technically impossible to do. The MTT test requires the formation of crystals of pharmacan from MTT and their subsequent solubilization (DMSO, isopropanol). The suspension cells themselves in the wells of the plate will distort the optical density, which is measured by the plate reader as a result of dissolution of the crystals of pharmacan.

Hello Lucía Noboa

Which cancer cell line you will be using? What is the culture medium? If the medium in question has almost the same composition as DMEM then I suggest you can go ahead co-culturing the cancer cells with mesenchymal cells. If not, then as mentioned by Norman Hafner you will have to adapt the cancer cells gradually to DMEM. During this gradual switch over to DMEM do check the cancer cells for morphological changes and other similar changes.

Best Wishes.

Shanmugapriya Shanmuga Sundaram

If these are primary cells of 2 to 5 days in culture, then it could be the cell debris. Looking at your image I doubt it could be bacterial contamination.

Probably if you are very sure about the contamination, I mean if you see the movement of bacteria, then the best option is to change the antibiotic from P/S to either gentamycin or Amphicilin, wash it with antibiotic PBS solution twice, and incubate it with media containing GENTA or AMPHI. This way you can slowly reduce the bacterial load and will be able to rescue your cells. Also include amphotericin B as antimycotic.

There is evidence of cross-contamination using LN2, this happens when the LN2 chamber is overfilled. This can be avoided by maintaining the appropriate LN2 level i.e., the cryo boxes inside the LN2 are in the vapor phase without getting in contact with the liquid LN2.

Cheers

Pia Nyeng you should maintain cells at between 30-80% confluency. Any less will induce a lag phase as the cells adjust to their surroundings. Any higher will induce a stationary phase as cells run out of resources and space.

My recommendation would be to allow your cells to grow to 80% confluency, and passage at a 1:8 ratio. This gives you three cell cycles for growth (2, 4, 8). If you know how long your cells take to divide, you can multiple this by three and come up with a protocol as to when to passage your cells.

I hope this helps!

Dear Begüm Kurt ,

Thank you for your contributions to my question. It has developed my point of view. (like always) I wish good look to you too, it was nice to be your colleague :)

Hi , I ended up freezing down DF-1 in 90% FBS + 10% DMSO and reviving them in DMEM containing 10% FBS and 4 mM glutamine (or glutaMAX) without Pen-strep at 39 C. Once the cells become confluent the medium could be replenished to contain Pen-strep starting from passage 2 or 3. You can also refer to our recent publication at

I hope it helps!

Best

Dear researchers, I'm trying to find the NP69 cell line, but ATCC and Sigma Aldrich don't have it anymore.

Can you suggest where I can buy them, please?

Thanks in advance Raffaele

Hello Dr.Stalin! In our laboratory we promote their adhesion by addition of phorbol 12-myristate 13-acetate (PMA) at a concentration of 100 ng/ml. This conducts the differentation of THP-1 monocytes into macrophages. However, when you say "without changing its natural I do not know if you mean to preserve their monocyte phenotype". THP-1 cells are non-adherent, so all treatments you perform to promote their attachment are going to have an impact on them. For instance, PMA tends to upregulate the expression of some genes in differentiated macrophages, which could affect the gene expression induced by other stimuli. If I am not wrong and I remember well, the treatment enhances inflammatory genes through NFKB pathway. In our case, as we are studying inflammation, we added a period of arrest, leaving the cells without PMA in order to reduce that possible enhanced expression.

I would recommend you (depending on the experiments you have to perform) to try different concentrations of PMA in order to add the minimum necessary to promote their adhesion without enhancing the gene expression. Firstly, I would check the expression of inflammatory genes after the treatment and after the arrest, to see how the treatment affected your cells and whether the arrest decreased the inflammatory response. Lastly, I would characterize the cells through flow cytometry or gene expression of monocyte/macrophage markers to see the profile of your cells.

Dear Sir Murtaza Ali , I have gone through the PDF file you attached with the reply to the question asked. What should I do, if I am dealing with a voucher specimen. For example: I collected live insects (very small size: about 400 micro meter) and store directly in 500ul TriZol. Should I crush samples in the same TriZol or take out and crush in liq.nit.

The protocol of TriZol requires measuring the sample size to use initial quantity of TriZol. I am unable to measure the number of cells in a full grown adult insect (very small size).

Dear Amit Kumar Shrivastava and Amanda Rocha can also answer from their experiences.

Thanks.

Hi! I would like to know how are your experience with MEG-01 cell line. I'm also working with it. Which technique do you use to characterise the cells? I'm using flow cytometry and my cells do not express CD61 and GPIIb/IIIa and I don't know why.

I have expierence with closed culture system only for delivery culture cells, I help a coworker that need to transport the lived cells. Then I used the system for some hypoxia study. maybe you can find some information about required volume of gas on hypoxia article.

Regarding study effects of closed systems I think that you can find something on web i don't understand if you need this on primary cells, for clinical purpose or something else.

For example, I can't download the article but you search something like this?

Riken Cell Bank, Tsukuba, Japan

You could try the following, if you want to increase your cell density:

hi

By an air traveler, you can transfer the cell to your laboratory immediately after defrizzing and placed in a 15 ml falcon's tube include complete culture medium for 12 to 24 hours.

good luck

It depends if you are fixing the cells for cellular markers or keeping them viable for live cell imaging markers. Also, whether you want to measure expression induction markers (mRNA expression), or on-going cellular processes (proteins, subcellular organelle activity like mitochondria and lysosomes).

Hello Catherine,

Don't use Collagenase. You can try to use trypsin/EDTA to separate cells from the collagen gel. I am regularly using the trypsin/EDTA solution. It works perfectly well.

A temp. of 4°C for collecting cell supernatants is not necessary. It may reduce activity of enzymes in your sample. It may help to reduce possible quality-reducing influencing during harvesting when this process needs much time or when room temp. is higher that 23°C. However, this (RT) has no fundamental influence on your sample quality.

In any case, if samples were collected at same conditions (4°C or RT or what else), results are comparable among each other. And this is the most important point in your experimental setting.

Hi there,

Why don't you just grow the cells in 5 cm and 10 cm cell culture dishes, wadh the cells and lyse them? Afterwards you can determine protein concentration and you know exactly how much protein you get...

Good luck,

Sebastian

Thanks for sharing this info.

Dear Jean Raynell Bello

Cancer cells are immortal. Therefore, their use is unsuitable for assessing biocompatibility. According to the international standard, fibroblast cells are used for this task such as L929, SNL and etc. All biological tests must conduct according to ISO standards 10,993–5:1999 (Biological evaluation of medical devices; Part 5: tests for in vitro cytotoxicity). You can read the following articles:

Flexible magnetic polyurethane/Fe2O3 nanoparticles as organic-inorganic nanocomposites for biomedical applications: properties and cell behavior

Preparation and evaluation of polyurethane/cellulose nanowhisker bimodal foam nanocomposites for osteogenic differentiation of hMSCs

The main problem with freezing is ice crystal formation - the DMSO added to freezing medium helps to prevent that. It acts as a cryoprotectant. Without the DMSO, chances are good your cells will be damaged as ice crystals form both outside the cells and inside. If you're looking at whole tumors, by guess is that you'll have some live cells left in the center of the tumor, but experience significant cell death in the outer layer of tissue.

If you only need to store overnight, why not just store them in a refrigerator? It'll keep them cold, but not freeze them to death, and thus hopefully leave you with more cells to work with.

I would like to add that the serum proteins also serve as substrates for Trypsin protease (an enzyme). Therefore, depletion of exosomes from FBS is not likely to alter the outcome. Whether an exosome preparation alone can "inactivate" the enzyme may depend on the source of the preparation and the number of exosomes.

Hello

I agree with Radhwan Nidal Alzidan; Theoretically speaking, your idea might work.

Following article may be helpful for you

Thank you Habib Rezanejad. I will check it out.

It is correct to use a gradient cooling down to cryopreserve cells, but it is best to thaw the cells in 37C as fast as possible.

Hi Erwin, ITR is just functional when packaging AAV. If you just want to express a gene in cells, ITR would have no effect, no need to delete it.

You could find more information about AAV on this website: www.genemedi.net/i/aav-packaging

Hi Monika Nanda,

I also have experienced such round shaped objects during primary culture of mouse BAs. In my case, usually such round drops were disappeared with completion of differentiation. I was also confused as like as you about contamination, but when I asked two professors (those are very experienced with adipose cell culture) they concluded that it's just dead cells and may happens in primary culture. So, according to my experience I think it's okay with ur culture.

In addition, as Matías Gabrielli already mentioned earlier about mycoplasma contamination, I would like to suggest u to check your cells for mycoplasma contamination.

Good luck!!!