Cell Line - Science topic

Thanks for your answer

Thank you both for your recommendations. I reached out to AcceGen regarding cell lines and examined the Neuromics lung CAF cell line. While it is indeed suitable for our purposes, it comes at a considerable cost. My concern lies in its limited expansion potential, typically spanning only 3-5 passages.

Hello,

I would use the medium that the supplier you bought the Caco-2 cell line from recommend, e.g. ATCC or ECACC, see file and link

Basically, if you're publishing in any Nature journal you're obligated to make cell lines available to other qualified researchers (either via public repository or by sending them yourself), either for free or for a "reasonable" handling cost. There might be some extra hoops to jump through if the PI is from a non-profit institution / university and you're trying to acquire them at a for profit company. Also, a lot of fluorescent proteins are patented by private companies, so any use of them by for-profit entities will probably require extra licensing agreements with those companies, even if the cell line itself was created by a non-profit institution.

I think your experience with acquiring said cell line is going to be more related to the specific parties and individual PI involved than the journal publisher.

Are you not running any proper house-keeping/reference genes for normalization?

Using RT-qPCR to validate CRISPR-based knockout (KO) experiments can be highly effective but may also encounter several challenges that need careful consideration and troubleshooting. Here’s a structured approach to effectively use RT-qPCR for confirming gene knockouts and addressing potential issues:

1. Design of RT-qPCR Assays

2. RNA Extraction and Quality Control

3. cDNA Synthesis

4. qPCR Reaction Setup

5. Data Analysis

6. Troubleshooting Common Issues

Conclusion

Using RT-qPCR to validate CRISPR knockouts involves careful experimental design, precise execution, and thorough data analysis. By addressing each step with meticulous detail—from RNA extraction to data analysis—you can robustly validate the success of CRISPR-mediated gene knockouts and gain insights into the genetic manipulations within your experimental system.

Perhaps this protocol list can give us more information to help solve the problem.

Creating a cell line that stably expresses truncated proteins involves several precise steps, from the initial molecular cloning to the establishment and validation of the cell line. Here is a detailed approach on how to achieve this effectively:

This approach provides a robust framework for generating a stable cell line expressing truncated proteins. It combines molecular biology techniques with cell culture procedures to ensure the production of a reliable and useful cell line for research and biotechnological applications.

Take a look at this protocol list; it could assist in understanding and solving the problem.

I've finally figured out the answer: the problem was in the electrophoresis equipment: the buffer was leaking and therefore the gel wasn't fully covered. I write this down in case other people have a similar problem, to check this I had to keep an eye on the mAmpere of the machinery (lower than 35 mA but higher than 20 mA).

I thank everyone who has been interested.

High fluorescence in flow cytometry for an Annexin V assay typically indicates a high level of phosphatidylserine exposure on the outer surface of cells, suggesting early stages of apoptosis or cell death

Hello Jean-Gregoire Marin

I also have in culture the TE.354.T cell line supplied by ATCC.

I maintained these cells in DMEM supplemented with 20% FCS and antibiotics (Penicilin/Streptomycine) according to ATCC.

I don't supplemented with L-Glutamine.

Like you, the cells I have take longer to grow than the time indicated by the ATCC.

I've tried to improve proliferation with other growth agents ((FGF-2, 50ng/ml) and (SDF-1, 100 ng/mL)) but without any real success unfortunately...

I hope it works out better for you.

In the meantime, have you been able to improve your cell growth?

Thanks in advance for any informations and/or suggestions

Best regards

Thank you Sir, Bruno Salomone Gonzalez de Castejon, we changed the method and got positive results. We still used the HEK 293 cells but with the PEI 40K reagent this time.

Michael Marino As for the vector size, the recombinated plasmid is same size with either donor plasmid or backbone plasmid, around 7kbp.

Thank you for your suggestion on RT-qPCR. I will check the virus product sufficiency.

Sure! IEC6 is a commonly used cell line in biomedical research, particularly in the field of gastrointestinal physiology and pathology. The IEC6 cell line was derived from normal rat small intestine epithelium. It was originally isolated and characterized by Quaroni and Isselbacher in 1982.

IEC6 cells have been extensively studied due to their ability to proliferate rapidly and form monolayers in culture, making them a valuable tool for investigating various aspects of intestinal cell biology, including epithelial barrier function, nutrient absorption, and mucosal immunity.

These cells retain many characteristics of normal intestinal epithelial cells, including the expression of markers such as villin and sucrase-isomaltase, which are indicative of differentiation into absorptive enterocytes. Additionally, IEC6 cells have been shown to express various transporters and receptors involved in nutrient uptake and signaling pathways relevant to intestinal function.

Researchers commonly use IEC6 cells to study intestinal physiology, epithelial barrier integrity, responses to pathogens and toxins, as well as to investigate the effects of dietary compounds and pharmaceutical agents on intestinal health. Furthermore, the ability to culture IEC6 cells in vitro provides a convenient model system for studying molecular mechanisms underlying gastrointestinal diseases such as inflammatory bowel disease, celiac disease, and colorectal cancer, among others.

A cell line that does not express both iNOS (inducible nitric oxide synthase) and CD163 (cluster of differentiation 163) could be a cell line derived from a tissue or cell type that does not typically express these markers under normal conditions.For example, many cancer cell lines, such as HeLa cells or MCF-7 cells, may not express iNOS or CD163 unless they are specifically induced to do so under experimental conditions. Additionally, certain immortalized cell lines derived from non-immune tissues, such as fibroblasts or epithelial cells, may not express these markers.However, it's important to note that the expression of iNOS and CD163 can vary depending on the experimental conditions and the specific context in which the cells are studied. Therefore, it's always a good idea to confirm the expression profile of a cell line using experimental techniques such as immunostaining, Western blotting, or flow cytometry.

It is difficult to have an single optimal way for every cell lines, each cells have their own strength driving multiple signaling pathway, upper limit of the plasmid copy received and interaction between GOI and its own proteins, hence affecting gene expression facility. We had tried at least four different transfection method with more than ten different reagents just to make a cell population to express 0.1% of GOI, while in 293T the expression was almost hitting the upper limit of detection in flow cytometry. Therefore, to generate an optimized protocol for each cell lines or primary cells are considered easier then to generate a single protocol for universal use. The cloning process or to achieve a single double KI/KO clone by CRISPR is very time consuming and troublesome, if you want to go through CRISPR, try look at the newest technique such as base/prime editing by Dr. David Liu. In the other hand, lentivirus is not, in our lab, lentivirus is an useful tool for large scale cell line production, a single production process may grant us enough amount of lentiviral particle for more then 100 times of usage. However, if a gene is already being difficult to express in such cell lines, I would rather look into an optimization of DNA construct, a small twist such as codon optimization or driving promoter sometimes grant you more then 50-fold of difference, that will be far practical then transfection process.

Best

Dear Dr. Anil Kumar Gupta

The article attached below will be helpful. Refer to Fig 1.

Regards,

Malcolm Nobre

Dear B R Siddharth

sincerelly i ever used the commercial cells since the number of lentiviral clones that we produced were limited, but since they are providing it as chemically competent i do not see any reason why you cannot expand and produce your chemically competent stock. Probably you will obtain cells with lower level of competency (more close to 10^6 cfu/ug than the 10^8cfu/ug and is it possible that you have to optimize more your ligation fo obtain some colonies during your cloning.

good luck

Manuele

If you have planned maintenance on your power supply, having a battery powered incubator at hand could facilitate the time before it comes back on. For people in similar situations, check out the Cellbox devices

Hello Pavithra,

Immortalized cell lines, in theory, can be passaged indefinitely. However, using cells at high passage number is not recommended since they may experience phenotypic and population changes due to genetic instability and selection pressures over time.

So, cell passage should be limited to prevent population and genetic drift.

There is no defined maximum number of passages for a cell line, but it is highly recommended to keep passage number low for any cell-based assay for reliable results.

Generally, passage number should be limited to no more than 15-25 for cell-based assays.

Best.

I was using Geltrex, based on the thin-layer method, for muscle satellite cells - also for the differentiation stage, and it worked really well.

Hi Malcolm Nobre

Many thanks for your answer. I have misunderstood the C33A cell line as non-cancerous cell line. I need instead any of the non-cancerous cervical cell line.

Hello Abdul-Rauf Nyewneh Nuhu

If you are planning to study the growth inhibition of HepG2 cells in vitro by using different concentrations of the drug, then MTT assay may be used because the MTT assay is commonly used to assess the cellular cytotoxicity caused by drugs (especially anticancer drugs).

Best.

Bei Zhong

I would say no problem at all, if any concerns, adapt Jurkat cell first in INS-1 cells complete growth medium (or vice-versa, however, I do recommend adapt Jurkat cells) to evaluate experiment condition and if secreted cytokines amount had changed upon treatment. Jurkat is not as attaching surface dependent as INS-1, therefore, if cell states and cytokines secreted had not change significantly after aforementioned process, then you're good to go.

Best.

@Malcolm Nobre thank you for your response. Yes, I preserved the cells via Mr.frosty at -80, and also carried out the quick thaw process. But I didn't maintain a temperature log for the -80 freezer

Hello Ayushi Gupta

Many anticancer regimens include compounds that bind to and/or modifies DNA. Fundamentally, drugs interact with DNA through two different ways, covalent and/or non-covalent modes. Covalent binders act as alkylating agents as they alkylate the nucleotides of DNA, while the non-covalent binders interact by three different ways: (i) intercalation, (ii) groove binding, and (iii) external binding (on the outside of the helix).

So, you could use various techniques to study the compound-DNA interactions such as electrophoresis, spectroscopy, mass spectrometry, viscosity measurements (viscometry), atomic force microscopy (AFM) and circular dichroism, and the effects of such interactions in the structure of DNA, providing some insights about the mechanism of drug action.

There are some references which I have attached that will help you to design your experiment without using cells.

Best.

Hi Henrike,

You blocked the antigens with donkey serum so in the control group, the secondary antibody won't have non-specific bindings. However, this did not rule out the possibility that the primary antibodies have non-specific bindings. Is it possible to use siRNA or certain approaches to deplete the target protein as a control?

Hope this helps.

Yuning

This might be a stupid question but is your new cell line from the same species?

Hi Prisicila, I have just read your comment from 2023. I bought the same cells and used the same conditions. I observed low growth during the first 3 weeks more or less. It is important to cultivate them in small flasks at the beginning, be patient and centrifuge them at least at 1000 rpm to obtain a precipitate before changing the flask

Hello Adila Zulkifli,

No, you need not wash the cells with PBS. Since LADMAC cells is a suspension culture with some loosely adherent cells, you may collect the cell suspension in the centrifuge tube (attached cells may be removed by tapping the sides of the flask until cells are dispersed), and centrifuge the cells at 1200 - 1500rpm for 5 minutes. Discard the supernatant, tap the cell pellet and resuspened the cells in fresh growth media at 1 - 2 x 10^5 viable cells/mL into a new flask.

LADMAC cells secrete CSF-1. CSF-1 is capable of supporting the in vitro proliferation of mouse bone marrow macrophages. If you wish to collect the conditioned medium you may follow the steps given below.

1. Allow cells to become confluent.

2. After 5 to 7 days, collect supernatant by centrifuglng at 125 x g for 5 to 10 minutes.

3. Filter using 0.2um filter.

4. Store aliquots at –20°C.

Best.

Tumor xenograft models play a crucial role in cancer research, allowing researchers to study tumor biology, evaluate therapeutic interventions, and investigate tumor-host interactions. NOD/SCID (Non-Obese Diabetic/Severe Combined Immunodeficiency) and NSG (NOD-scid IL2Rγnull) mice are commonly used immunodeficient mouse strains for establishing tumor xenografts due to their severely compromised immune systems, which allows for better engraftment of human tumor cells. Here's a comparison of tumor xenografts in NOD/SCID and NSG mice:

Overall, both NOD/SCID and NSG mice are valuable tools for tumor xenograft studies, but NSG mice offer advantages in terms of higher engraftment efficiency and a more severely compromised immune system, which may better recapitulate the human tumor microenvironment in certain contexts. The choice between these models depends on specific research objectives and experimental requirements.

Hello Santosh,

the Jurkat E6.1 grow even well without sodium pyruvate, I did not observe any difference in my experience.

Regards

Yes, Good Luck!

Despite being called a "clonal" cell line, after multiple passages, you end up with a drift that generates a mosaic culture of cells that are different in their osteogenic differentiation potential. You could "re-clone" your line to identify clones that still have a good osteogenic potential: Get 30-40 single clones, expand, freeze, differentiate each clone with usual GP and vit C when confluent, and select/use only clone(s) that can differentiate and mineralize.

Hello Zc Liu

The K562 cell line expresses very low levels of class I antigens, and specific viral gene products suppress class I expression in K562 cells. This type of repressor effect in K562 demonstrates the site of action to be transcriptional.

Agents like IFN-g inhibits RNA synthesis that suppress class I transcription and favors the accumulation of positive regulatory factor thus allowing increased HLA class I transcription. This clearly demonstrates the post-transcriptional control mechanisms for HLA class I gene expression. This type of mechanism is seen not only in K562 cells but also in other tumor cells as well as in undifferentiated embryonic tissues. So, it may be concluded that both positive and negative regulatory factors are involved in transcriptional regulation of major histocompatibility complex class I genes in K562 leukemia cells. For more information you may want to refer to the articles attached below.

Best.

Dear Tina Trinh ,

I would highly recommend to get in tough with the company asap, since they are stating this at the end of the link you have provided:

"Due to the sensitive nature of primary cells and cell lines, all quality related issues about the cells products must be reported back to us within ONE month period after receiving the products, no quality warranty (i.e. replacement of cells) will be provided after the ONE-Month period. Thank you for your understanding."

They should check their batch of the cell stock and give you maybe more clear thawing protocol.

We are usually thawing (different cells) like this:

Thaw the cells 30-60s with in a 37°C water bath (ice should/must still be visible which will keep the cells still cool). Than we are are transferring the cells into 10 ml pre-warmed medium I do use a pipet for that set to 800 µl. After 3-4 pipetting steps adding warm medium into the vial and removing than cells an the medium into the 10 ml reservoir, I spin the cells down (5 min 250 x g) aspirate the medium and plate the cells in new medium into the cell flask or dish. On the next day the medium is exchanged to get ridge of the rest of the DMSO.

Best wishes

Soenke

Hi,

Thanks for your responses.

Yes, these cells are grown in L15 media with 10% FBS, with P/S, in an incubator without CO2. I don't observe any changes in media colour/transparency, and the cells seem to grow normally, although a bit slowly (I think they are still adjusting to being thawed).

I will inoculate some culture on LB tonight to know for sure.

Hello Mansur Dabai Salisu

You may consider the following reasons.

1. The choice of which cell type to use for transfection is a critical factor that is often overlooked. Since each cell type is likely to respond differently to a given transfection reagent, choosing an appropriate cell type is necessary to maximize results. For instance, some cell types like MCF7 or HepG2, prefer to grow in clumps or clusters which is not ideal for transfection because minimal membrane surface is exposed which compromises uptake. Blood or immune cells that lack the proper endocytic machinery can minimize uptake and there are the macrophages that have an evolved uptake mechanism, but quickly breakdown and destroy endosomal contents. So, select the right cell line for transfection.

2. Make sure that the cells are not 100% confluent (should be about 60-80% confluent) or in stationary phase at the time of transfection because actively dividing cells take up foreign nucleic acid better than quiescent cells.

3. Most cell types should be used between 4 and 25 passages for optimal transfection.

4. There could be a possibility that the DNA concentration may be too low, or DNA may be degraded. In such a case, you may increase the ratio of DNA :transfection reagent, and you may confirm DNA integrity by A260/A280 spectrophotometer reading (should be at least 1.7).

5. You may use serum-free media for DNA dilution because some serum proteins may interfere with complex formation. You may also increase complexing reaction incubation time.

6. Test the culture for contamination.

7. Do not use antibiotics at the time of transfection because cationic lipid reagents increase cell permeability. As a result they may also increase the amount of antibiotics delivered into the cells, causing cytotoxicity and low transfection efficiency.

8. Cells could suffer mechanical damage during the experimental steps. So, do not vortex or spin cells for extended period.

9. Some transfection reagents could be compromised due to improper storage.

Best.

okay. thank you very much.

following your recommendation, I am going to try make a kill curve.

thank you.

Dear Colleague,

Thank you for your inquiry about the application of CRISPR/Cas9 technology in MDA-MB-231 and MCF-7 cell lines. The use of CRISPR/Cas9 for genome editing has indeed been widely adopted across various cell lines, including those relevant to breast cancer research, such as MDA-MB-231 and MCF-7. The success of CRISPR/Cas9 in these cell lines is well documented in scientific literature, underscoring its potential for understanding cancer biology and developing therapeutic strategies.

MDA-MB-231, a triple-negative breast cancer cell line, and MCF-7, an estrogen receptor-positive line, have been utilized in numerous studies to investigate the role of specific genes in cancer progression, metastasis, drug resistance, and other crucial aspects of cancer biology. CRISPR/Cas9 has been employed in these contexts to knock out genes of interest, introduce specific mutations, or modulate gene expression, thereby elucidating their functions in cancer development and progression.

Several key factors contribute to the success of CRISPR/Cas9 in these cell lines:

Literature reports demonstrate successful applications of CRISPR/Cas9 in these cell lines, ranging from basic research on gene function to more applied studies on drug resistance mechanisms and potential therapeutic targets. These studies underscore the adaptability and effectiveness of CRISPR/Cas9 in manipulating the genomes of MDA-MB-231 and MCF-7 cells for diverse research purposes.

In conclusion, CRISPR/Cas9 genome editing has been successfully applied in MDA-MB-231 and MCF-7 cell lines, contributing significantly to breast cancer research. The continued refinement of CRISPR/Cas9 methodologies and an increasing understanding of cell line-specific responses to genome editing will further enhance the utility of these cell models in cancer research.

Best regards,

Reviewing the protocols listed here may offer further guidance in addressing this issue

Performing a proliferation assay with Ki-67 in GFP+ Jurkat cells is a valuable approach to study cell cycle dynamics. However, the issue you’ve encountered with GFP loss during the permeabilization step is common. Let’s address this problem and explore potential solutions:

Remember that optimizing protocols often involves trial and error. Be patient, systematically test different conditions, and document your findings. With persistence, you can achieve reliable GFP and Ki-67 staining without compromising GFP signal.

Ok let me try. thank you

Hello Kiana Florentino,

In vivo models for investigation of antidiabetic activity of bioactive substances always have the problem of long experimental cycle, complicated experiment, and high cost. Compared with the animal models, cell culture could observe the experimental results much more simply, economically, quickly, and objectively. 

You may want to refer to the article attached below which provides different types of in vitro diabetic cell model.

The cell lines may be obtained from

ATCC

or

ECACC

Good Luck!

it could be that the 293T cells may have more robust antiviral responses compared to Jurkat and Raji cells, leading to the inhibition of TTV replication. This could include the activation of interferon response pathways or the expression of restriction factors that limit viral replication.

Another thing you should investigate if there is any nutrient deficiency in the culture medium. Have you check the other culture medias as well ?

Didier Poncet We did use NaoH and NaCl for denaturing the DNA fragments in the gel before transfer. We have taken enough care while setting up the transfer so that there is no direct contact between the paper towels and reservoir buffer. Thank you.

Yes. You would come to know whether there is a problem in freezing or storage because HepG2 cells would have been much happier to be stored in liquid nitrogen than in - 80 deg C for a period of nearly 5 years which is a long duration.

Having frozen 2 × 10^6 cells, I would have expected a reasonable number of cells in the selected field in the image. You should have obtained at least 85- 90% viability after thawing to get most of the area covered with cells which I do not observe in this case.

Best.

Hello Eyal Goldfarb

You may use HEK293 cells (human origin)

1. Easily growing, attached cell line.

Yes.

2. Easily grown on regular DMEM media.

Yes.

3. The cell must not have any endogenous current.

Not as efficient as CHO.

You may want to refer to the articles attached below for more information.

Best.

This might be the paper you are looking for:

Dear Esteemed Colleague,

Thank you for your inquiry regarding the efficiency and quality of the NEB Q5® Site-Directed Mutagenesis Kit. The Q5® Site-Directed Mutagenesis Kit is designed by New England Biolabs (NEB) to facilitate the rapid and precise introduction of mutations into double-stranded plasmid DNA, leveraging the high-fidelity Q5 DNA polymerase. Below, I provide a detailed evaluation of this kit based on its features, application performance, and user feedback, which should aid in assessing its suitability for your research needs.

The NEB Q5® Site-Directed Mutagenesis Kit employs a robust, streamlined protocol for generating point mutations, insertions, or deletions within plasmid DNA. A key component of the kit is the high-fidelity Q5 DNA polymerase, which ensures accurate amplification with minimal error rates.

The NEB Q5® Site-Directed Mutagenesis Kit stands out for its high efficiency, fidelity, and ease of use, making it a highly regarded tool in the field of molecular biology for precise genome editing. Its comprehensive design, accommodating a wide range of mutagenesis applications, along with positive user feedback, underscores its utility and effectiveness.

Should you require further insights or have specific questions regarding the application of the NEB Q5® Site-Directed Mutagenesis Kit in your research, please do not hesitate to contact me.

Best regards in your research endeavors.

📷

You

The difference between 293T and Phoenix cells 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

ChatGPT

Dear Esteemed Colleague,

Thank you for your inquiry regarding the differences between 293T cells and Phoenix cells. Both cell lines are instrumental in biomedical research, particularly in the fields of gene therapy and vaccine development, due to their utility in producing viral vectors. Below, I provide a detailed comparison highlighting the key distinctions and applications of each cell line.

In summary, the choice between 293T and Phoenix cells depends on the specific requirements of your viral vector production. 293T cells offer versatility and high efficiency for various vector types, including lentiviruses, while Phoenix cells are tailored for streamlined retroviral vector production. Understanding these distinctions will aid in selecting the most appropriate cell line for your research endeavors.

Should you require further clarification or wish to discuss your cell line selection in more detail, please do not hesitate to contact me.

Best regards in your scientific pursuits.

Take a look at this protocol list; it could assist in understanding and solving the problem.

Hi Henriette,

were you able to get hold of the cells?

If yes, are you able to share them with us?

Thank you,

Georgios

There could be many reasons. But, we need more information pertaining to your specific experiment.

Is the host protein of interest endogenous or exogenous? Presumably the viral protein is expressed exogenously using transfection or viral transduction. If both proteins are being supplied exogenously, you may be running into an expression issue in HFF1 cells. Be sure that both exogenous proteins are being expressed in the HFF1 cells. If the host protein is endogenous, the HFF1 cells may simply not express it to a high level naturally. You can measure expression of the proteins using western blot of your input lysates. If you are not getting good expression of exogenous constructs in HFF1, then you may turn to alternative methods of supplying the trans-gene (like viral transduction).

HEK-293-T have a high transfection efficiency, so you may be getting more of both proteins expressed and are therefore able to see an interaction. Generally, an interaction in HEK-293-T cells is considered biologically relevant. That being said, these cells lack many components that other cell lines have.

Aside from this technical consideration, there are many other reasons an interaction may occur in one cell line and not another. Different cell lines will have different global protein expression as well as post-translational modifications (PTMs). So, if your host protein is endogenous, check baseline levels in both cell lines. Also check for differences in the banding pattern between cell lines to spot PTMs. For instance, the endogenous host protein may be cleaved or phosphorylated in HFF1.

As an additional consideration, cell lines may respond differently to the method of trans-gene delivery. For instance, the HFF1 cells may have more intact innate immune signaling pathways than the HEK-293-T cells, including ones for foreign DNA sensing. When you supply the trans-gene using transfection, the HFF1 cell components are able to identify the foreign DNA as a threat and carry out an inflammatory signaling pathway. This type of signaling can alter host proteins (via PTMs or gene expression/repression) and may lead to loss of a protein-protein interaction in that cell type.

Did you change the media or the cells itself?

You can extract RNA from the cell lines (treated group and control) and then do qPCR for the expression of your three genes of interest. For your natural products use a chlorofom:methanol:water extraction and use a rotary evaporator to dry before resuspending in a small volume of DMSO. You can then add a volume of that to your cell culture media. As for how good your cell line will be for modelling cervical softening, I cannot tell. I advise using a positive control.

Hello Chandra Kishore,

You may use density gradient centrifugation. In density gradient centrifugation depending on the density of the particles in the sample, similar substances will group together when exposed to rotational force. Because dead cells and cellular debris are fractured, they become less dense than living, healthy cells. Adding in certain separation reagents such as Ficoll can purify the sample by acting as a barrier that only one population can pass through.

For instance, in a 50 mL centrifuge tube, you may layer 18ml of your cell suspension onto 12ml of a Ficoll-paque combination. Centrifuge your tube for 15 minutes at 400x g. When centrifugation is complete, you will note that the live cells will collect at the interface and the dead ones will form a pellet at the bottom of the tube.

Another simple method as mentioned by Samir would include centrifuging the cell suspension at 150-200g for 10 mins. You may discard the supernatant which consists of cell debris and resuspend the cell pellet in fresh medium.

Best.

Thanks for your kindly suggestion ！I will try. The lane 7 is protein from lung（mouse），and 8 is spleen.

I would need to know what genes you had knocked out to make the cell line tumorigenic. It might be possible your cell line hit the Hayflick limit.

Yes, works great!

It's very strange that under these conditions your DAUDI cells are not growing. Even if the cells are not properly growing, I would suggest to change the medium at least once a week; and I would increase the FBS to 20% to see whether this increases the growth rate, if it does, you can later on again reduce it to 10%. As long as the cells are not proliferating, I would not split them at all, just change the medium.

Are your cells still viable and just not growing or already dead?

Did you test the cells for mycoplasma contamination?

Can you post a picture of your cells?

Thank you so much for your advice and references! It's very helpful.

Erika Avila

I met the same problem! Have you figured this out?

fy

If the dispersed cloudy layer doesn't show any bacterial cells, it could potentially be cryopreserved cells that have surpassed their storage lifespan. Typically, if these are dead cells, they might follow the movement of the shaken plate. However, if they are bacteria, they usually exhibit flexible movement even when the plate is moved in a specific direction. Dead cells would often appear as shattered cell debris.

Dear Prof. Najla Saeed Dar-Odeh

Thank you for the above important question. After going through the literature, I would advise you not to use Hep-2 cell line for your research in laryngeal cancer because Hep-2 is in fact comprised of cervical adenocarcinoma cells, derived via HeLa cell line contamination. This was shown by Stan Gartler in 1966 and published in 1968. Later this cell line was subsequently confirmed via PCR to be positive for human papillomavirus DNA sequences.

For more information you may want to refer the following articles attached below.

You may use either SNU-1066 or RK33 cell lines.

Please refer to the paper attached below.

Best Wishes.

Have you done three replicates of the same experiment and obtained the same results? Are the levels of the housekeeping gene similar in all the WB?

@Malcom Nobre.

Dear Dr Nobre I checked the process of cell freezing and figured out that the -80 cold room had been out of order for a day. I think the problem was related to cold room.

When target-control is done, 2^-(ΔΔCt) is used.

Hello Xu Weifeng

No, macrophage cell lines cannot be used to study how bacteria inhibit the host complement system. The reason is as follows.

Neutrophils provide a front line of defense against bacterial infection. They capture and destroy the invading bacteria by setting traps and ingesting them.

The antimicrobial capacity of neutrophils is higher compared with that of macrophages. The neutrophils are equipped with a huge assortment of microbicidal mechanisms and use multiple antimicrobial molecules stored in enormous amounts in granules sequentially formed during granulopoiesis. Production of ROS is most prominent in neutrophils as compared with macrophages. Following phagocytosis, neutrophils use the “crosstalk” between oxidants and granule proteins to attack ingested microbes in a collaborative way. Several antimicrobial proteins that are an important part of the neutrophil arsenal are lacking or scarce in the tissue macrophage. These are defensins and cathelicidins, the major families of mammalian antimicrobial peptides of neutrophils, and lactoferrin.

Macrophages, on the other hand, are distributed in tissues throughout the body such as intestinal macrophages in the gut, Kupffer cells in the liver or the microglial cells in the brain. A macrophage begins life as a monocyte, but upon infection, monocytes are rapidly recruited to the tissue, and once within inflammatory lesions, monocyte receptors are bound by cytokines, antigens, and other stimuli, which rapidly activate the maturation of monocytes into macrophages.

Though the use by mammals of a system with two dedicated phagocytic cells, neutrophils and macrophages, working cooperatively represents an advantageous innate immune attack strategy, macrophages and neutrophils are not replaceable. Each one has its own importance.

You may want to refer to the article attached below for more information.

Best.

Hello Kar Wei Chin

Lentiviral vectors are less genotoxic and represent a safer option for genetic engineering, but the efficiency of lentiviral transduction is inefficient for NK cells and has remained variable requiring multiple rounds of transduction and/or post transduction cell enrichment to achieve acceptable transgene expression. So, lentiviral transduction may be the method of choice for genetic engineering when it comes to safety for therapeutic applications. Moreover, lentiviral vectors can incorporate transgenes into the genome of non-dividing cells.

On the other hand, retroviral transduction requires active cell division. Therefore, retroviral transduction may be utilized with NK cell lines such as NK-92, which you will be working on, that have continuous and unlimited proliferation capacity. Moreover, higher transduction of expanded NK cells have been reported in several studies with retroviral vectors.

You may want to refer to the articles attached below for more information.

Best.

The slow growth and poor adherence of U-87-MG cells could be due to several factors. Here are some potential reasons and solutions for the issues you are encountering: Thawing and passaging procedures can affect cell viability. Ensure that the U-87-MG cells are properly thawed, and the viability is assessed using a trypan blue exclusion assay or other viability assays. Consider obtaining a new vial of U-87-MG cells from a reliable source to rule out issues with the cell quality. Even though suitable media are being used, there may be variations in the culture conditions that affect U-87-MG cells differently than other cell lines. Double-check the pH, osmolality, and the presence of necessary growth factors in the media. Additionally, confirm that the CO2 concentration and temperature in the incubator are appropriate for U-87-MG cells. Consider optimizing the trypsinization time and concentration for U-87-MG cells. Some cell lines, including U-87-MG, may require a specific substrate coating for proper adhesion. Consider using poly-L-lysine or other suitable coatings to promote adherence.

Yes there is another cell line in the same incubator. There is no adhesion or reproduction in that cell line in the same way. Thank you for your reply. I will change the brand of FBS and try it.

Thank you very much! Yes, this is on our list (shared below) but unfortunately the only cell line out of these that we've been able to find available for purchase is MDA-PCa-2b. Do you know where we can get access to the cell line you mentioned?