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When I isolate RNA from cultured cancer cells and check for different epithelial (E Cadherin, CK19, Klf5)and mesenchymal (Snail, Twist, Zeb1, Vimentin etc) markers by qPCR, somehow the trend of expression of these markers is never consistent. Does this happen? Is there any reason?
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Srishti Kashyap I did not go deeper into it since I dropped the EMT thingy for my project! So couldn't solve it. This is my opinion/observation: If your cells are obviously epithelial or mesenchymal, as in if you see the difference in the phenotype of the cells when you culture them, then you will surely see consistent results for the EMT markers (atleast some markers). But if your cells look similar phenotypically, then you might not see consistency in the makers expressed. This is just based on my observations, no concrete data to prove this! Hope it helps.
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Goodnight
Sorry to bother in this Sunday
But I would like to ask a few questions:
Does anyone know about how to induce the generation of polyploid giant cancer cells (PGCCs) in melanomas with "targeted drugs"?
How to measure the presence of polyploidy in the tumor?
How to induce the generation of giant polyploid cancer cells (PGCCs) in melanomas?
Is there any protocol to induce the generation of polyploid giant cancer cells (PGCCs) in melanomas?
Is there any way to study the immortalization and regeneration of tumors with cellular and molecular biology? Is there any protocol?
Best regards,
Matheus Correia Casotti
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Scientists have developed a new method that can easily create immortal human mammary epithelial cells. The cells could greatly facilitate the examination of cell immortalization as it actually occurs during cancer progression. Every day, some of your cells stop dividing, and that's a good thing.
I am sharing a pdf, it would be very helpful for you.
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I am treating some cancer cells to see if they are directly linked to centrosomes. I did immunostains on gamma-tubulin, flow on cell population after treatments, and qpcr on some centrosome-related genes. I am just wondering what other methods I can use to show that my drug or particular active ingredients affect cell centrosomes. Thanks.
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Maybe looking at kinase activity might show another functional layer close to the actual phenotype. With our technology, you could measure differentially between a treated and untreated sample, and look at differences in up or down regulated kinases and its signalling pathways. You could outsource this research to our in house facility. If you like to learn more you can always investigate on our website, and reach out to me at evbreemen@pamgene.com.
Maybe this is worth a shot!
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Is there any in vitro assay in order to prove if the cancer cells are undergoing hypoxic condition , which is not based any quantification of protein or RNA?
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Try Agilent Seahorse. We have a couple of that.
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As NCCS Pune has stopped to supply these cell lines, I am not able to get them.
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I am also struggling to find a Jurkat E 6.1 cell line; the cell line we received from NCCS was contaminated. I cant able revive it. If you still have these cell lines, please help me.@
I appreciate any help you can provide.
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In the previous MTT assay, it was confirmed that the survival rate was decreased, and in the DAPI experiment, an increase in the apoptotic body of the substance-treated group was confirmed.
After that, it was confirmed that there was more blank space as the cell viability decreased in the substance-treated group by treating the cancer cells with a substance.
However, as you can see, the cleaved-PARP, which should be increased, came out more strongly in the control, and also the bax decreased compared to the control. Have you ever had a case like this?
* The top line is total .
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Make sure the reagent you are using is not reducing the tetrazolium. Many times this control is missing when starting to test a new drug and the results are misleading. Also, the MTT assay measures mitochondrial activity, you may try trypan blue or propidium iodide to make sure your treatment is increasing cell death. Dose- and time-response experiments are essential to establish the best assay conditions. If you saw many fragmented and pyknotic nuclei in your DAPI experiments, look for other caspase-dependent and -independent apoptosis players.
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Nano-medicines are widely used in Oncology but are their actions on specific target cancer cells or they act on nirman cells also?
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Nanomedicine can provide a targeted drug delivery system that would be ideal for cancer treatment. It is a system which releases the drug at a preselected bio site in a controlled manner. Nanoparticle (NP)-based drug delivery systems have been shown to have many advantages in cancer treatment such as, good pharmacokinetics (PK), precise targeting of tumor cells, reduction of side effects thereby making a significant impact on cancer treatment.
Specially designed nanoparticles deliver medicines like chemotherapy straight to the tumor. They don't release the medicine until they reach it. This stops the drugs from damaging healthy tissues around the tumor.
The small size of nanoparticles allows them to deliver medicines into areas of the body that would normally be hard to reach. One example is the blood-brain barrier, which prevents toxic substances from getting into the brain. It also blocks some medicines. Nanoparticles are small enough to cross this barrier, which makes them a useful treatment for brain cancer.
By incorporating small-molecule drug conjugates (SMDCs) and miniaturized biologic drug conjugates (mBDCs), including peptide–drug conjugates into controlled-release polymeric NPs for multistep delivery to tumors, it could be possible to combine both the superior PK and tumor accumulation of NPs with deep penetration and specific tumor cell targeting of released SMDCs and mBDCs for optimal targeted cancer therapy.
So, cancer nanomedicine could usher in a new era of successful targeted therapy.
Best.
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I want to ask you because the morphology of the HSC-3 cells I grow is different from the general shape. If the cells look like this, what's the problem?
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Your cells have a more fibroblast-like morphology when compared to the image information provided by the Japanese cell bank, JCRB.
I too find it unusual and doubtful that it is HSC-3.
Correspondingly, the
You should start up the cells in another storage tube.
After all, if they have a similar morphology.
HLA type, SNPs, STS, and other genomic markers should be analyzed to verify if it is HSC-3.
Alternatively, karyotyping would also be useful.
The cause of the change in cell morphology could be
1. mistaken for another cell line.
2. cross-contamination with another cell line
3. not using the recommended media or culture equipment.
I will consider these factors.
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I am trying to do ATAC seq for my epigenetic research on human prostate cancer cells ( PC3 and LNCaP). I have a bottle of genomic lysis buffer. I am not sure if I can use it for ATAC seq. I would appreciate anyone answering me or suggesting me the right buffer. Thank you.
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No, you cannot use genomic lysis buffer for ATAC seq.
ATAC seq is used to assay the open chromatin regions in their native conformation. So, you need to use mild detergents in the ATAC lysis buffer which will help solubilize the cellular membrane but permeabilize the nuclear membrane. This will allow the transposition reagents to enter the nucleus and act on the chromatin in its native state. The ATAC lysis buffer will ensure that the majority of the cell membrane is lysed while preventing lysis of the nuclear membrane.
If you use the genomic lysis buffer, there is a probability that the nuclear membrane will be lysed resulting in the loss of native chromatin conformation as well as it will cause leakage of the nuclear contents.
The ATAC lysis buffer contains non-ionic detergents like digitonin, NP-40 and Tween-20. You may refer to the protocol given in the links below.
An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues - PMC (nih.gov)
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I'm doing apoptosis assay using the flow cytometer on cancer cells (mcf-7). I'm wondering what's the ideal event run count and speed to be used?
Thanks,
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You can go with 10000 events and if you have more cells you may go up to 40000-50000 cells events count , keeping same for all. Speed wise - medium or High should work as you are collecting the data in log scale.
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Can i used the commet assay to differentiate between the cancer and normal cell?
how commet assay result i interpret to distinguish that this is the normal cell and this is cancerous cell?
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Hello Himanshu ...,
Very simple and short answer.....Commet assay is not an appropriate assay to differentiate between normal and cancerous cell. Use other specific reliable methods to differentiate between normal and cancerous cell.
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I am trying to figure out through what means Acetoacetate may be converted to acetate naturally within a healthy, or cancerous cell (though specifically non-hepatic cells). I am especially interested in any methods which involve the TCA cycle. I have been currently considering acetoacetate converting to acetyl phosphate then acetyl phosphate to acetate within the mitochondria; however, I do not know if this is a possible pathway. Any information upon potential pathways for this conversion, or how viable the acetyl phosphate pathway is would be greatly appreciated.
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Google "acetoacetate degradation" : With CoA, acetoacetate can be converted to 2 acetyl-CoA, which enter the TCA cycle for further degradation.
e.g. "Ketone body utilization. β-Hydroxybutyrate and acetoacetate are interconverted by 3-hydroxybutyrate dehydrogenase (3HBD) according to the intracellular redox situation. Acetoacetate is then activated to its coenzyme A (CoA) ester by succinyl-CoA: 3-oxoacid CoA transferase (SCOT). Thiolytic cleavage of acetoacetyl-CoA to acetyl-CoA is catalyzed by acetyl-CoA acetyltransferase 1 (gene ACAT1), also called 2-methylacetoacetyl-CoA thiolase (MAT), which got its name from its role in isoleucine degradation where 2-methylacetoacetyl-coenzyme A is its substrate."
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Hello, I am reading a paper saying their compound can lead to apoptosis of HepG2 cells; however, it came to me that is it possible that the cytotoxicity of the compound causes the apoptosis? Or what’s the difference between cell cytotoxicity and cell apoptosis?
Let’s say, If one compound is capable of leading to cancer cell apoptosis, how about its function on normal cells? Can it cause the apoptosis of the normal cell too?
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Hello Li Qingyun,
Apoptosis is a form of programmed cell death that has important roles in development, aging, and disease. Apoptosis is initiated by a tightly regulated signaling cascade that results in caspase activation. Several features characterize apoptosis, including cell shrinkage, membrane blebbing, chromosome condensation, nuclear fragmentation, DNA laddering, and the eventual engulfment of the cell by phagosomes. Two major pathways are operated during apoptosis. The intrinsic cell death pathway is governed by the Bcl-2 family of proteins, which regulate commitment to cell death through the mitochondria while Activation of the extrinsic cell death pathway occurs following the binding on the cell surface of “death receptors” to their corresponding ligands such as Fas, TNFR1, or TRAIL.
On the other hand, cytotoxicity is the degree to which a substance can cause damage to a cell. A substance or process that causes cell damage or death is referred to as cytotoxic. Cells that are exposed to cytotoxic compounds may undergo necrosis (uncontrolled cell death), apoptosis (programmed cell death), autophagy, or stop actively growing and dividing to decrease cell proliferation.
Drug induced mild insult could induced apoptosis, while high level of insult or sustained high level of insult may induced cytotoxicity. The drug-mediated cytotoxicity may operate several death pathways including apoptosis, necroptosis, necrosis etc depending on the level of insult generated by the experimental drugs.
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I got an experimental result where the mitochondrial transmembrane potential of drug-treated cancer cells has increased than untreated cells, as well as ROS generation, was found to be higher in treated cells. The experiment has been repeated multiple times and a similar result was found. The experiment was based on JC-1 and rhodamine-123 staining method.
Thanking you in anticipation for your answer and suggestion.
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Hello Rajesh Kumar Meher,
Yes, you are right. Drugs-mediated increase of mitochondrial transmembrane potential could induce apoptosis in treated cancer cells in vitro. The altered mitochondria membrane potential (MMP) may result in calcium exflux in cytosol. Depletion of Ca2+ level from internal stores like mitochondria and decreased replenishment cause ROS generation and at the same time elevated cytosolic free Ca2+ may further generate cellular ROS. Thus, increased MMP, cytosolic free Ca2+ and ROS levels could induce apoptosis in cancer cells in vitro.
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Hi everyone
Has anyone ever expanded a cancerous cell type in serum free medium ?
I am trying to see approximately how long the cells, irrespective of their type, might survive without serum in their media?
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It likely is highly dependent on the cell line you are testing. It's also important to consider the difference between cells surviving and cells growing in serum-free conditions.
The environmental demands of cancer cells are not necessarily much different from their somatic counterparts. The key difference is that cancer cells (1) grow when they should not and/or (2) do not die when they should.
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on a tight budget with little experience in studying cancer in mice which method should we pursue for successful implatation of MCF-7 cancer cells on mice
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The most recommended method is to use estradiol pellet to support growth of MCF7 cells, but implanting cells with materigel can work (though low tumor formation rate) which is more cost effective.
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I am now conducting research on HER2-positive and HER2-negative breast cancer cell lines. I'm looking to confirm the overexpression of HER2 receptors in HER2-positive cells versus HER2-negative cells. Could you possibly assist me in determining how I may accomplish this either quantitatively or qualitatively?
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You may carry out immunocytochemical (ICC) staining of HER2 receptor as the expression of this receptor in the cell lines that you have by constructing a CMA (cell microarray) containing the breast cancer cell lines with the goal of rapidly screening for antigen of interest (HER2) in a relatively easy, rapid and cost-effective manner.
Please refer to the research article attached below. See Materials and Methods on Page 1598 for the protocol.
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Can anyone tell me how to stimulate pancreatic cancer cells for cytokine release in cell culture ?
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Hello,
I hope you are doing well. Please take a look at this link ( ).
Bests,
Pooya
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Im going to plan a cancer study. Im appriciate to read and answer my question.
1. Using NCI-N87 gastric cancer cells, i'll induce subcutaneous tumor in mouse.
But between flank and hind limb, i cant choose the region for cell injection.
Which one is better between them?
2. Im also wonder the necrosis in growing tumor tissue. i want to take MRI imagies and tumor vascular histologies. How can i take care of the tumor mass to prevent necrosis?
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Tumor cells are usually subcutaneously implanted on the flank or dorsum of animals (mostly mice or rats), in which the transplanted cells proliferate and form measurable tumors.
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Suggestions and recommendations are welcome. A protocol used and experience would also be helpful.
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Simpler and easy methods are using bright field microscope and flow cytotoxicity. u can check out these papers:
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It is an experiment to check the effect of a certain phytoestrogen on a particular cancer cell proliferation in-vitro.
The results consists of
Six absorption readings from control group
Six absorption readings from X phytoestrogen at concentration A
Six absorption readings from Y phytoestrogen at concentration a
Six absorption readings from Y phytoestrogen at concentration b
Six absorption readings from Y phytoestrogen at concentration c
THANK YOU.
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Hello John Kurian,
Since you are testing two phytoestrogens (X and Y), out of which Y phytoestrogen has three concentrations (a,b,c). So you have to see the effects of two phytoestrogens, therefore it would be better to use two-way ANOVA followed by post hoc analysis (to compare any two groups). Actually, in your experiment, you have first factor = X phytoestrogen; second factor =Y phytoestrogen and then interaction between these two factors X vs Y (third factor).
If you want to only analyze the effect of various concentrations (a,b,c) of Y phytoestrogen (one factor) , then one-way ANOVA followed by post hoc analysis would be appropriate.
Hope this helps
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I am new to culture HUVEC in 3D collagen on a microfluidic chip. I recently met a problem.
The problem is that the HUVECs will die after being cultured in 3D collagen for 2 days. in addition, A549 lung cancer cells also die when culturing for 3 or 4 days. In fact, the cells are seeded onto the surface of collagen lumen. However, the cells remained in inlets/oulets and PDMS channels live well.
Therefore, I think the reason may be the shortage of nutrients for cells in lumen.
I have no one to ask so I want to get some help from you guys.
any suggestion is welcome.
Thanks
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I am also meet the similar problem.
I embed cells in the 3D collagen, but they die.
Have you solved the problem? Does mix FBS with collagen work?
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I am looking for a method to assess the function of cancer cells and macrophage co-culture. rather than inducing the macrophage to either only M1 or M2, I would like to see what is the effect of coculture between a spectrum of macrophage and the cancer cell in vitro. I think this can recapitulate more the in vivo microenvironment where both M1 and M2 macrophages co-exist.
I am using both the J774 and THP-1 cell lines. May I know if there is some well-established protocol/ published article that tried inducing the differentiation of both M1 and M2 at the same time?
Thanks!
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I think CCL7 is one such chemokine that can induce both M1 and M2 in-vitro. I am not sure there might be more. Check this article - Macrophage Polarization: Different Gene Signatures in M1(LPS+) vs. Classically and M2(LPS–) vs. Alternatively Activated Macrophages
But if this has not been shown already, perhaps you would first like to validate protocol and then verify the mixed phenotype obtained.
I agree that its more valuable to study the spectrum in case of macrophages. Did you tried inducing them separately and later mixing for experiments ? You could also sort the mixed population to work with.
Good luck!
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Articles remark that cells plated at the right density have enhanced efficiency of organoid culture establishment. Plating too densely will result in increased cell death at the core of the dome.
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Dear Melis.
I have established CRC organoids even with 2x105 cells per 20 uL in a 12-well plate (usually 5-6 domes of 20 uL matrigel). If we go for 1000 cells per 20 ul it will be millions of plates and a lot of matrigel, as normally we get resection tissue and we collect around 1x106 cells after digestion. In fact, larger density is better as shown in this link (at least for mouse organoids).
Remember to put the plate up-side-down during polymerizing the matrigel to ensure that the cells stay in the top of the dome. There is no doubt that low cell density is not good either, so try to titrate your cells in different densities and evaluate the growth rate and diameter.
Good luck.
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As my cancer cells have undergone quite amount of passaging , thus with every passage , there are stress factors which either float around them or are stuck on the cells, as a result my cells are not geting the growth media , i have tried everything by filtering the media, increasing the antibiotic concertration and also lowering FBS but the stress factors are still present , i have been changing their growth media in every 12 hours now , is there any other way to make them go back .Please share your view
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From your content it seems that something is wrong with the cultured cells. Its important to find out whether its really stress causing deterioration of culture conditions. If yes, then try to determine which kind of stress factor i.e. physical (temperature, osmolarity or pH) or chemical (chemical composition of culture medium, drugs etc) are involved in cellular stress. Then what kind of stress is being generated (free radicals or hypoxia)?
Once you are confirmed the actual triggering factor of the stress, then its very easy to target in order to reduce it. If you find out that the oxidants are generated and causing deterioration in culture conditions, then you can use the appropriate "antioxidant" to scavenge the free radical or oxidants under in vitro culture conditions.
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Looking for a fast-track journal to publish one of my papers in the field of image processing for non-hodgkin leukemia cancer cells detection method.
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What about irradiate nanoparticles as a metal like ZnO or Iron oxide to treat cancer cell.
Any suggestions about the techniques of the exposure and the dose that kills 50% of cells
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thank you
I want in details irradiate ZnO NPs by Neutron activation
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We have been using the Celigo to count and measure the size of breast cancer cell mammospheres and we are having issues getting accurate results. The machine tends to break up large mammospheres into 3-4 pieces and count them individually, while also counting a bunch of smaller individual mammospheres as 1, and also counting things that are just single cells near each other.
I have played with the settings numerous times and have worked with a technical person from Nexcelom and cannot get the settings to work for us. I was wondering if anyone had any luck using the Celigo for these purposes? I have tried analyzing the mammospheres using the Colony setting and the Tumorsphere setting.
Thank you in advance for any insight.
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So I will preface this by saying I work for a completely different company just so that is clear but I've run into a few groups with a similar problem and it seems like the software itself needs some adjustments. This is likely a back end fix/code rewrite that Nexcelom and companies like it need to do, to make the systems work more effectively across a variety of assays and cell types.
On that note Jennifer, depending on how often you and your team run these assays (mammospheres, colony, organoid, etc) there are other systems which are more dedicated to the task are doing (ie counting and sizing), the GelCount being the one I know. Not suggesting this is a fix for your problem but just a different solution. All the best.
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I am doing the NO assays using griess reagents without kit, please tell me what will be working concentration of griess reagents and how the can be done spectrophotometrically. Thanks
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Hello Noor Akbar
You can use 100μL of supernatant in a microtiter plate well. Add 100μL vanadium (III) chloride (8mg/ml) to each well (for reduction of nitrate to nitrite, since the Griess reaction is specific for nitrite) and this is followed by addition of the Griess reagents (50μL sulfanilamide (2%) in 5% phosphoric acid and 50μL N-(1-Naphthyl) ethylendiamine dihydrochloride (0.1%) in distilled water).
After 10-20 mins incubation at 37 degree C (protect from light), absorbance is read at 540 nm. Concentration of NOx in the samples is determined from the sodium nitrite standard curve. You need to prepare a standard curve for serially diluted nitrite concentrations.
Please note: Use of colorless media would be appropriate because any color in your samples may give you erroneous absorbance readings.
Please refer to the article below for more information on the protocol.
Best Wishes.
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Could anyone recommend a good antibody to check BOK expression levels in Immunofluorescence (mainly cancer cells)?
Thanks a lot!
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Thanks for the suggestion!
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TGF-β and BMP signaling are present in the cancerous cell show almost the same function of chemoresistivity, enhanced proliferation, higher EMT transition. Then why would cells have both mechanisms? Also, what is the major difference between both of them?
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I think the best way to look at it would be to ask "what is the function of those two pathways in a normal cell of that tissue?", if you are able to figure that out, you maybe able to answer the next question which is "what are these activated two pathways doing in a cancer cell?". In normal condition a lot of genes have similar tasks, because their functions are critical to cell survival and health. "Redundancy" is the norm, rather than the exception. On other hand, and to specifically address your question, to figure out why the two pathways, which have identical end points are both activated, you will need to go back and dissect the pathways components looking for a common denominator which is probably receiving an upward signal and from it it converges on both pathways. Check out a diagram of the RAS gene, which shows you that an activated RAS converge on both pathways ...bRAF and PI3K pathways, thus activating them. In this instance, as in yours, the end result will be upregulated nuclear transcription!.
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I'm trying to understand the mechanism of L-amino acid oxidases (LAAO) on cancer cells and healthy/normal cells. Why can LAAO treatment induce apoptosis efficiently with a low dosage on cancer cells compared to healthy cells? Can you guys help me with clarification on this problem?
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I would like to recommend the following paper:
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Hi all, I have done mouse subQ injection with cancer cells. But with same cell numbers on each mouse, I got so much variation with tumor volume. How can I minimize the size variation? Many thanks!
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This is a bit late but I was searching for an answer to my question and saw this. I get loads of variation as well, this is normal for tumours, I've asked other PhD students who work on different cancer cell lines, we all have gigantic error bars for the growth and variation between groups.
For myself, I've also noticed if I implant earlier passages (about 1-2 weeks after thawing/below passage 10) versus later passages (>4 weeks later/above passage 12), I get significantly larger tumors. I believe it's just got to do with artificial selection for those that proliferate better, thus, they grow better, after all, they have high mutational burdens, wouldn't be surprised if passage 12 versus passage 5 aren't identical clones.
To summarise,
1) Nothing to do with injection/mice moving
2) My tumour growth and a previous students' is completely opposite, I'm unable to get the same results (he had reduction for a B cell-deficient strain, I find no difference)
3) Tumours are weird
Now, I really need to figure out how to get absolute numbers for TILs, so if you have an idea of how to back-calculate from % (most accurate method), I would be grateful.
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Could someone kindly clarify why aerobic glycolysis is required for cancer cells? That is, if tumor cells used OXPHOS, would they be unable to grow and metastasize as well?
The most commonly cited benefit of aerobic glycolysis seems to be accelerated glucose uptake, but why is this required for long-term growth as opposed to short-term spurts? The timeline for cancer progression is normally months or years, not hours or days.
Moreover, despite consuming glucose faster, total ATP production from aerobic glycolysis is lower. That is, during the time OXPHOS consumes 1 glucose molecule, aerobic glycolysis may consume 13 glucose molecules -- yet this yields fewer ATP molecules.
If correct, total energy production inadequately explains the relationship between cancer cells and aerobic glycolysis.
Is the timing of ATP essential? Perhaps cancer cells need fewer shots of ATP molecules but at a higher frequency rather than wait for one large batch from OXPHOS?
Or what are the other benefits of aerobic glycolysis over OXPHOS?
Ultimately, why would cancer cells be less successful with OXPHOS?
Thanks in advance for your help.
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Aerobic glycolysis creates an acidic micro environment that compromises the TEJ of the normal native vasculature creating vasogenic edema which increases the glucose concentration in the tumor micro environment, favoring unregulated cellular proliferation. MREPT can detect vasogenic edema enabling minimally invasive ablation of cancer before cellular mass inhibits diffusion, and much before the formation of tumor vasculature can create “washout “.
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Good Day I don't know if you guys will read these but I really need an advice.
So here's the thing I compiled all the authors available from various databases about the anticancer property of Peperomia against multiple cancer cell lines.
These are the sample data that I got from multiple papers
Peperomia
Author 1
Roots > LC50 > against various cancer cells (including cell viablity)
Leaves > LC50 > against various cancer cells (including cell viablity)
Stem > LC50 > against various cancer cells (including cell viablity)
Author 2
Roots > LC50 > against various cancer cells (including cell viablity)
Leaves > LC50 > against various cancer cells (including cell viablity)
Stem > LC50 > against various cancer cells (including cell viablity)
Author 3...
Author 89.....
What would be a good way to represent my data in my paper? Is there any statistical tool or analysis that I can use?
Thank you sincerely!
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Hello Brent Randolph . Tomas Koltai's suggestion is good for visual presentation. A table will also suffice ordered according to plant part. Alternatively it can be ordered by cancer type (l.e. colorectal cancer, assuming not too much variation in type of colon cancer celline i mean if it was epithelial versus mesenchymal you might want to list those separately) then list and cite evidence for each plant part accordingly. If two or more references conducted the experiment in same plant part on the same cancer cell line it is best to present the concentration (if variable) as a range if it is too widely spaced in value (ex. 150ug and 250ug) or an average if it is too narrow, ex. 20ug and 25ug. Hope this helps.
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We are planning to use Anti-Cisplatin modified DNA antibody for fluorescent microscopic imaging of cancer cells. In this regard, availability of specific protocols for staining cells with such antibodies is required.
Suggestions are highly solicited.
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Dr.Ali I did go through the datasheet provided with the antibody but nothing was mentioned in detail with regard to immunofluorescence.
Thank you.
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We know that the brain sends and directs meaningful messages to control the patient's cells.
as we know, The brain is affected by factors such as diseases And we know that the brain also controls other organs of the body.nevertheless,Damage to the CELLS is visible on eeg?
Is Cancer Effective In EEG?
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Stomach cancer is cancer that may affect any part of the stomach and extend to the esophagus or small intestine, and it causes the death of nearly one million people annually. It is more prevalent in Korea, Japan, England and South America. It is more prevalent among men than women. It is associated with eating too much salt, smoking, and also low intake of fruits and vegetables. Therefore, it is believed that its spread in countries such as Korea and Japan is due to the consumption of salted fish mainly by Koreans and Japanese, as well as the use of canned food and food preservatives. Mucosal colonization of H. pylori is believed to be the main risk factor in about 80% of stomach cancers
Stomach cancer is diagnosed through an endoscopic examination that allows a biopsy to be extracted from the affected tissue, and then analyzed to confirm the presence of a tumor. Dr. Riccardo Rosati, a specialist in gastroenterology at San Raffaele Hospital in Milan, says, "Before undergoing treatment, the patient needs to do a series of other ultrasound and other examinations to check the areas, glands and organs covered by the disease, in order to determine the degree of its progression.
As a researcher, I believe that stomach cancer cells do not send messages to the brain due to the lack of associated neurons
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are there conditions to choose the cancer cell type to test a new nanoparticle-drug conjugate on it?
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Saurabh Mandal Marwa Abd El Kader Zaater Malcolm Nobre the nanoparticle has a molecule that interacts with the lipid membrane itself of the cancer cell. so basically, any cell can be used i think.
However, what i want to know is why in some papers the authors work on breast cancer cell lines and not colorectal cell lines for example? is it just a matter of preference (they have experience in dealing with this type of cells)? or is that because of the high fatality rate of breast cancer patients in the world? (this is just an example)
And thank you for your answers
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The dielectric constant of cancer cells has been obtained experimentally and has been stated in some articles at a certain wavelength. Is there a report on the refractive index of cancer cells, especially mcf-7 cell, at different wavelengths?
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Thank you
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I need this very much because I have no experience on it. So I want to publish an article by using this kind of web based tools.
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Use the above link, it is perfect to build a transcriptome profile either by commands for a certain cell line (with real RNA-seq data), or just using the user friendly website. It is also possible to address comparisons between cell lines by applying the proper ratios, like GeneA/GAPDH in cell line X vs GeneA/GAPDH in cell line Y, using FPKM or TMP values.
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There are many method to check about stemness of cells. But, I need a method that can compare stemness within few minutes. Is there any method ?
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Aldefluor is one of the fast method.
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Can we perform spheroid formation and invasion assay of cancer cells using Lionheart FX Automated Microscope?
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I don't know about the Lion Heart Imaging system, but I can advise about the CytoSMART Omni. We have developed that system in such a way that you can monitor any type of well plate for weeks, so this may be useful for your spheroid research.
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when designing a chimeric antigen receptor for cart t cell therapy can't we use a target which is also exist in T cells but express excessively in cancerous cells?
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I presume that you've found an answer to this question given all the recent publications in CAR T. To answer your question in short, yes, it should be possible, but why would you risk fratricide? Do you have a target in mind?
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I want to do some cell migration assays and I would like to know if it's possible to grow these cells in suspension
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Hi
Saifun Nahar
, we are cultivating ID8 cells with 10% FBS. It will not cause them any problem BUT! Different % of FBS can cause differences e.g in gene expression and other aspects. I would make sure to do ALL experiments with the same % of FBS so you will avoid differences between results. I hope it helped, have a nice day :)
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Hello everyone,
I'm studying about cancer cell and its pathways and here I have some questions. Do cancer cell still have their wild-type proteins or genes? or all of those wild-type proteins and genes already turned into mutant ?
any answer will help me a lot!
Thanks in advance!
-Richca
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Depending on the stage of cancer the percentage of mutations varies. But in any stage you won't get all the genes are mutated. It's the same for proteins.
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I wanted to buy an incubator in which we will be maintaining neuronal as well as cancer cells. With advancement in technologies, i was wondering whether air or water jacketed incubator is best for academic lab purposes? All your inputs are much appreciated. Thanks
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Richard Jayaraj Water has a larger heat capacity than air, so it's easier to keep the temperature in case of a power failure for some time. Otherwise, check the total cost each system will incur.
Depending on the humidity and air quality in your lab and facility, you might face the risk of mould contamination of the incubator. While copper linings and shelves are better for keeping contaminations at bay, stainless steel is easier to clean and disinfect in my experience, e.g. by fumigating with acetic acid (keep over the weekend with an open bowl of HOAc and set at 50 to 60degC).
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Hi all,
I was performing a coculture experiment of cancer cells along with CAFs and my goal was to look for the CCND1 gene expression (Hypothesised to go up in the coculture).
What I observed was that the uncocultured cells showed higher CCND1 expression compared to the cocultured ones.
Is this due to an effect observed due to high confluency of cells leading to contact inhibition and decrease in CCND1 level?
Appreciate any kind of output!
Thanks
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Hello,
There could be a synergy between cyclin D1 and p27 in their role of extracellular signaling. Both could serve as mediators for anti-mitotic stimuli throughout the cell cycle. In the presence of anti-mitotic signals (confluency), the cell can engage in a cell cycle exit programme, even before mitosis simply by preventing p27 degradation and diminishing levels of cyclin D1.
You could please refer to the research article attached below.
Best.
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Hello, I am trying to isolate single cell colonies form MCF-7 breast cancer cells. I didn"t get any colony in my plate. they were edited by CRISPR but the deletion does not affect their growth/survival. Thank you!
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Yes it will work fine, the detailed protocol can be obtained from this link. Best of Luck.
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I am using a lentiviral TET-ON system for conditional overexpression of my gene of interest in primary kidney cancer cells. I use viral load from transfection of HEK293T cells after 48 and 72 hours and perform selection for 2 weeks using Puromycin (using a concentration I have optimized for the cells I am using). After to weeks, I use Doxycycline at 2mg/ml (adding fresh media everyday) for 72 hrs. However, in western blots, I only see slight increase in expression of my gene of interest compared to -Dox. How can I increase the efficiency of overexpression?
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Did you say 2 mg/ml?
This is too much or you were trying to say 2 ug/ml?
Is the viral packaging system optimizied ? Try making another viral load with perhaps gfp to see if the viruses are viable in your system.
Or go for pcr to see if you have viral integration into your transduced cells. Doxocycline should work ..
You can also revise your cloning method again to see if anything is missing there.
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For the evaluation of Lysosomal destabilization in cancer cells, if there are two different drugs encasulated in NPs then how the effect of NPs containing two different drugs and their combination can be evaluated using Lysotracker red DND. If the two different NPs cause lysosomal destabilization in their individual treatment then the combination of both the NPs should cause impressive result like more lysosomal destabilization.
Then if the combination shows more lysosomal destabilization then what would the impact i.e. towards which phenomena it indicate or if lysosomal destabilization in a lesser fraction then wich phenomena will it indicate? Does it has any relation with autophagic flux?
The entire process can be evaluated by laser confocal microscpopy?
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To do this you could measure cytosolic cathepsin D level. If it increases > untreated cancer cells, you know that it has been damaged. You can also assess lysosomal disruption with a lysotracker. Good luck!
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As the title if the question indicates, can someone tell me what are the proteins that are overexpressed in HeLa cancer cells?
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Pradyumna Shejwalkar we were trying to know what the overexpressed are proteins in these cells so that we can predict the binding of these proteins to a peptide of interest
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These breast cancer cells, dissolved at -80 degrees, did not adhere to the T75 flask in the first 20 hours, what can be done?
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Hello
The cells may be under stress. Did you follow a proper thawing protocol. The cells have to be washed in order to get rid of the freezing medium containing DMSO.
Sometimes, if the cells are not frozen in a proper manner or if there is variation in storage temperature then there could either be loss in cell viability or change in cell behaviour as in this case. Have you checked the cell viability after thawing?
I suggest you culture these cells in a smaller surface area like 6-well plate or T-25 flask so that the cells may come closer to each other and begin to adhere and grow.
Good Luck.
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What might be the possible reason for granulation of ct26 cancer cell line?
I tested media(RPMI), serum and all of cell culture conditions but I cant get rid of granular cells.
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What does it appear, can you post a photograph?
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After collecting the cancer cells treated with anticancer substances, the centrifuge is rotated at 1300rpm for 30 seconds, and after removing the remaining pbs, 1*10^6/80ul LYSIS BUFFER is added based on the number of cells calculated. And (voltexing 1 minute, freezer 5 minutes) After repeating this process 4 times, it is rotated to 1300 centrifuge for 5 minutes, and the supernatant is collected and used as a Western protein. c parp shows a good trend, so apop seems to be concentration-dependent, but bax and bcl2 tend not to show well. The desired result is an increase in bax and a decrease in bcl2. What conditions would you like to change?
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Do you know which apoptosis pathway is activated by using chrysin? As suggested above try first to see which pathway is activated (Intrinsic/extrinsic pathway). If the intrinsic pathway is activated why do you expect to see changes in Bcl2 and/ or Bax levels? I think you have to analyse more Bcl2 familiy members and not only bcl2/bax. Remenber: If you induce intrinsic apoptosis where bax is involved, bax is often just going from the cytosol to mitochondria where it forms pores to release cytochrome C without any change in total bax level. This is eg driven by proapoptotic BH3 only proteins like Bim, Bid or Puma. Sometimes you just have to block antiapoptotic/proapoptotic interaction by using specific BH3 mimetic eg inhibiting BCL2/Xl/W or Mcl-1 to induce apoptosis without any change in protein levels. So analysing Bcl2 familiy protein levels not always answers your question. BTW I think the most important antiapoptotic proteins are BCl Xl and Mcl1. Bclxl overexpression or Bax/bak double knock outs will block intrinsic apoptosis. Please remember that caspase 3 activation by the intrinsic pathway can feed back to activate caspase-8.Hope this helps…
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Is that CCK-8 test can also react with DH5a (E.coli)?
In numerous studies have shown that CCK-8 or MTT tests for detecting the proliferation of cancer cells incubated with certain bacteria. However, in my recent work, I find that DH5a also presents a great influence on the OD450 of the CCK-8 test. I really wonder the validity that using this method for examining the proliferation of co-culture bacteria and cancer cells. Furthermore, I apply Hematocyte Counter for checking this result, I find that the so-called phenomenon of cancer cells proliferation have been definitely disappeared, or even displayed cells number decreasing. I did not know how to explain these conflicting results.
Many thanks for your kindly response.
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Kaili Fu I think it difficult to observe cell proliferation. Actually, I tend to consider that the coculture of bacteria and cells in vitro may be a competitive relationship. The CCK-8 assay, Edu kit, and Flow Cytometer always present the confusing result, because the live bacteria can also react to these reagents. I did not try the LDH assay, but many species of bacteria also carry the LDH enzyme.
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I tried staining MCF7 and MB231 for Vimentin and Cytokeratin antibodies, and surprisingly, I saw both cell lines showed both markers. According to the literature, MB231 is a mesenchymal type cell and MCF7 is luminal, so shouldn't it be that MB231 shows more cells positive for Vimentin as compared to MCF7?
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Hi Rafał Watrowski . Adity Pore was just asking why markers for the hypothetical EMT do not make sense in the 2 cell lines she is looking at.
I agree. The EMT is just a hand waving explanation of how epithelial cells, that are normally sedentary, suddenly become motile and invasive when they are cancerous.
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Hello everyone,
I was wondering if anyone would be willing to give me a vial of a prostate cancer cell (PC3 or DU145) in the GTA area. I’m currently examining protein protein interactions but I think it may be cell line specific.
thanks in advance :)
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I am using JC-10 (Enzo life science) dye for quantify mitochondrial membrane potential in CRC with 10 uM final concentration. I treated cell with CCCP 10 uM for 20 min for a positive control. I took the reading for J-aggregate at Ex/Em: 490/525 nm and for monomeric JC-10 Ex/Em :490/590. However I could not get the appropriated 525/590 nm ratiometric decrease in MMP even in CCCP treated set. I also repeated the experiment applying JC-10 in serum free medium to stain the cell, and again I Couldn’t get the result. I have a doubt in my protocol of staining buffer. Many companies supplying JC-10 kit provide two types of assay buffer to stain the JC-10 in the cell. I could not find the information about the ingredients mixed in such assay buffers.
If you have any suggestion for me in staining buffer and in whole procedure. Please help
Thank you
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To read fluorescence in a microplate reader the clear bottom black or white plate is highly preferred. Request to your vendor for it once, it is available in the market by several companies.
If you could not find the plat, then you can use the general plate as well, however, it will have interference with neighboring wells too, which may affect your result output.
In my case, I use a black plate with clear bottom for the microplate reader.
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Hi all. I would appreciate any help regarding this. I am trying to grow patient derived primary bladder cancer cells from surgical samples using mechanical and enzymatic digestion approach. Initially cells were growing well but after few days, it seems the cells go into senescence stage. I have attached the corresponding images of the cells after 21 days. Any suggestions/comments on this would be very helpful.
Currently I am using DMEM(high glucose)-F12 media with 3% FBS + EGF-insulin- Cholera toxin-hydrocortisone as supplements.
Thanks and regards
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Hi, Sumit
Even cancer cells are not easily immortalized, especially human-derived cells.
From the attached photo, it seems that the stress caused by the subculturing induced cellular senescence.
In general, if you try to establish a cell line from about 10 cancer tissues, a few will probably be established as continuously culturable cell lines.
In order to establish cell lines more efficiently, immortalizing genes such as TERT, c-Myc, SV40T, HPVE6E7, etc. are often introduced. Of course, this may alter the properties of the cell line.
Perhaps using a hypoxic incubator to reduce oxidative stress may also be useful in extending cell life.
Best,
Best, Narumi
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Hi,
I have been doing some experiments to reveal cytotoxic/antiproliferative effects of the new compounds in the cancer cell lines. After 24 hr incubation with compounds in Hep3B cells, I realized a specific structure on the wells with different compounds even my positive control (adriamycin), but not on control wells. So I am attaching an image of the wellhole shaped structure. Can you give an idea what this structure is or looks like? Thank you
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Please see our paper for more info
Mutant p53s and chromosome 19 microRNA cluster overexpression regulate cancer testis antigen expression and cellular transformation in hepatocellular carcinoma | Scientific Reports (nature.com)
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I want to measure the cellular uptake of nanoparticles in cancer cells using ICP-AES. I want to know the concentrations of NP (subtoxic concentrations or high concentrations) and the incubation of NP with cancer cells ( short time or long time) and the protocol for cancer cell digestion before applying to metal analysis using ICP-AES measurements.
Thanks in advance.
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You can refer to the highlighted part of this article.
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Hi,
My lab team and I are planning on making a co-culture with mesenchymal cells and cancer cells. The thing is that mesenchymal cells were grown in DMEM, and according to the ATCC the cancer cells are supposed to be culture in RPMI. Is there any protocol that can help us determine how to adapt the cancer cells to DMEM in order to make the co-culture?
Any suggestions would be appreciated
Thank you!
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You may transition the cells into the new medium gradually. However, the metabolism of the cells will be changed. It is quite often that you may find different labs use different media to culture "the same" cell line. The question is whether the new medium may affect your study. Note that there are also many variations of DMEM (and RPMI too), although there is only one from ATCC. Even just changing from low glucose to high glucose in DMEM may have significant impact on the cell. There are a lot of differences in the formations between DMEM and RPMI (e.g. several folds differences in amino acid concentrations). So I would expect the cells would behave quite different after the transition.
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I have done MTT assay on cancerous cells for silver nanoparticles using different concentrations. I have the absorbance for the treated, control and blank. How do i calculate IC50 for my dose ?
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Dear Sakshi, thank you for asking this interesting technical question. In this context please have a look at the following article which might help you in your analysis:
Comparative Cytotoxicity Study of Silver Nanoparticles (AgNPs) in a Variety of Rainbow Trout Cell Lines (RTL-W1, RTH-149, RTG-2) and Primary Hepatocytes
This article has been posted on RG as public full text. Thus it can be freely downloaded as pdf file.
Also please see the following useful RG link:
Calculation of IC50: an easy way to calculate IC50 using MS EXCEL
Good luck with your work and please stay safe and healthy!
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I am looking for breast cancer cell line models that reflect the PAM50 HER2-enriched (HER2-E) classification, which may overlap, but not be necessarily the same as HER2+ breast cancer cells.
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The breast cancer cell lines representing the PAM50 HER2-enriched (HER2-E) are
1.HCC1954
2.HCC202
3.AU565
4.HCC1419
5.SK-BR-3
6.UACC-893
Please refer to the research article below for more information.
I hope I have answered your question.
Best Wishes.
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5-fluorouracil, oxaliplatin and irinotecan purchased from Sigma are failing to show an inhibitory effect on cancer cells (tested on HT29 and A549) in cell viability assay (MTT). Please advise on what could have gone wrong. They are not expired. I have tried preparing fresh solution as well-also did not work.
Please note that I am well-versed with MTT assays and have in the past worked extensively with 5-FU and other chemotherapeutic compounds, so I do not expect it to be a problem with a standard assay such as MTT.
Thank you for your help in advance!
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Just a suggestion. Why don't you thaw a new vial of HT29 and A549 each if you have a stock, and do the MTT assay again with 5-fluorouracil, oxaliplatin and irinotecan. I just wanted to know whether you have exposed these cell lines to the drugs before ?
Best Wishes.
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I am currently writing an introduction to my thesis and reading "Biology of Cancer" of Weinberg.
The book is so awesome and interesting, I just can not express how much I love it: it touches all themes of cancer, starting from "what is a cancer cell" and finishing with the biological points of its development/biology (nearly 1 000 pages).
I would like to write an intro to my thesis which would be a pleasure to read, and I would like to find an inspiration in the book, like the one about cancer. I don`t want reviews (already downloaded them), I would like scientific book, which would cover many aspects of telomeres and/or aging.
Could you recommend one, please?
Thank you very much for all suggestions.
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Soon I will be making a co-culture of cancer cells with mesenchymal cells. I have cultured the mesenchymal cells in DMEM for several weeks and they have responded fine, but I do not lnow if it is suitable for cancer cells. I do not have much experience working with animal cell culture, so any feedback is very welcome!
Thank you in advance!
Best wishes from Costa Rica,
Lucía
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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.
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Hi,
I am currently working on a project that involves characterizing FT190 and FT194 cell lines, but I can't find much information about them. I want to know as much as I can about them, including the patient's nationality from which they were derived from and if they are chemo and radiation naive. Thanks!
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These are immortalized FT secretory cells we developed a few years ago from women who had surgery for non-cancer indications.
A comprehensive characterization of these lines (WGS, RNA-Seq, scRNA-Seq, etc) will be submitted soon. The cells have been sent to ATCC for easier access. Some are already available on their website. Hope this helps.
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Recently, I am exploring the potential function of one gene in cancer. Since the TCGA datasets as well as some other clinical data show that this gene is up-regulated in cancer and especially in basal-like subtype. I tried to knockdown it by shRNA and the result show the depletion of this gene accelerate the proliferation, migration and invasion rate of cancer cells. I also tested the over-expression in cancer cells and the result show the over-expression impaired the proliferation rate. This observations is also consistent with some previous report which reported it as tumor suppressor. Although I found that expression of this gene maintain the basal-like feature of breast cancer. The results is still confused me.
My question is whether you know some similar works or papers.
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not unfortunately
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Cell culture.
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It is about 6-12 hours, it grows really fast. For me, A flask is fully confluent after 48 hours, even it is splitted at 1-5.
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Dear esteemed researchers,
I am looking for proteins from breast cancer cell line MCF-7 to study docking of my proposed organic compounds. Please guide me about the most significant proteins from MCF-7 which should be used to study docking interaction of organic compounds.
Thanking you
Sachin
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Thank you Lili Lin
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I am hoping to identify the proteins present in different areas of primary cancer cells (cytoplasm, nucleus, membrane etc.) by mass spectrometry. However, my access to fresh cells is limited, therefore I am hoping to carry out this experiment on frozen primary cells. The ProteoExtract Subcellular Proteome Extraction kit looks promising and is compatible with frozen cell pellets. But I am finding it difficult to find research papers that used this technique to analyze frozen primary cells and not cell lines. If anyone has any experience in this area, I would greatly appreciate some guidance? Thank you!
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if the cells were snap frozen in PBS, you can fractionate them with little loss and at least separate the nuclei, cytoplasm and cytoskeleton and, if you wish, also mitochondria (with lost activity) or even ribosomes. Use the standard procedure from Sigma or Biorad protocols.
If the cells have been incubated in any lysis buffer, especially with detergents, your chances of a good separation are not that great.
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Need this number to do some mathematical differentiation, appericiate it very much!
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Please refer to the link below for more details on BT-474 breast cancer cell line.
Additionally, I would like to mention that the doubling time for BT-474 has been quoted differently by different sources.
Doubling Time for BT-474 = 84 hours (PubMed=9671407),
78 hours (PubMed=25984343)
I hope this is helpful.
Best Wishes.
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My research is to analyse the estrogenic activity of possible substances that migrate from plastics using the E-screen test based on the ability of MCF-7 cells to proliferate in the presence of oestrogens. However, so far I have not been able to validate the assay since I have failed to see significant differences between the effects caused by my positive control (17β oestradiol at almost any concentration) and my negative control. There is one paper that suggest that different "sublines" of MCF 7 cells show different sensitivities to oestrogen. So, I was wondering if you have been able to see differences between the proliferation in the presence of E2 and in presence of the negative control. and where did you get your mother stock of MCF-7
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It may be the presence or absense but above all I want to make sure it is like this. The rest could be sensitivity
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Nowadays i make recombinant protein. but i can`t see single band. alwasys i see multiband in E.coli system (in reducing system but multiband). but when I overexpressed that protein in cancer cells, I always see single band (this time I use myc or flag tag)
his tag problem?? or just expression species problem?
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Hi there,
Are the tag localized at the same extrimity of the protein? If yes, and as Michael, I doubt the His tag is responsible for the fragility of the protein expressed in bacteria.
The protein might be sensitive to degradation in the bacterial environment whereas in cancer cell it is more stable... Do you extract the proteins prior analyses? If yes, are the protocols equivalent?
Do you detect protein by WB using anti tag antibodies?
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I am working on oral cancer cell line and i would like to observe the in vitro antioxidant activity in treated cancer cell line to evaluate the enzymatic activity like SOD, CAT, GST. I have read many papers and they have tried inducing oxidative stress in the cancer cells to observe this activity. I would like to know why we need to induce it when the cancer cells can also produce free radicals.
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