Questions related to Cell Line
I am currently working on SK-N-SH cell line, nevertheless they are growing too slow while tried both in DMEM+10%FBS and RPMI+10FBS%. I split them 1-4 and 1-2 in DMEM and RPMI accordingly but they differentiated.
If anyone is working on SK-N-SH, please let me know the best growth medium and suggestions.
I'm looking for the syngeneic cell line ENU1564 rat mammary adenocarcinoma cells. Does anyone have them, preferably in the USA, and are willing to share with me?
I have a colony formation assay. I am seeding cells in a 96-well plate. The cell line is NTERA, 150 cells per well. I am seeding the cells with DMEM fortified with 10% FBS. Each well will have 200uL of this media. I will be incubating the treated cells for 14 days. I will be counting colonies on days 4, 7, 10, and 14.
How often should I remove the old media and replenish the wells with fresh media?
Thanks in advance.
Hello Dear all!
I would like to know if it is possible to have a stable cell line with a response element attached to a luciferase signal, also, Could it be possible to have in the same stable cell line a fluorescent protein that indicates the insertion within the genome of the before mentioned response elements/luciferase at the moment of generating it? (Have in mind that the plasmid used to generate the stable cell line confers a hygromycin B resistance to the cell line, after selection with this antibiotic)
if that is the case would it mean that when looking at the cells in a fluorescent microscope with a suitable laser exciting the fluorescent protein all the cells should exhibit fluorescence?
Thanks in advance for your help
Hi everyone. I am trying to set up an MTT assay with VCap cell line. I have previously given it a try based on a few research papers but I had a huge variation every time. Can anyone please help me with the number of cells to be used and the procedure to obtain consistent results?
It is the first time I want to grow this cell line. I thawed the cry-tube which was in nitrogen tank and then try to aspirate it with proper medium.The cells was not aspirated completely. Can you help me please?
I am using IMR-32 cells as a neuroblastoma cell line model for MYCN amplified cells. This cell line has poor transfection efficiency. Using lipofectamine 3000 reagents, I am getting only about 30% efficiency for transfection. I am looking forward to the suggestions for increasing the transfection efficiency of IMR-32 to a higher-end.
Hi. We have been observing this strange cell death morphology during months. Cells die and acquire a "fried egg" morphology as it is shown in picture (A431 cell line). When it happens, all the cells die alike. It appears randomly in cell plates and not in all the experiments, but we are thinking that affected-wells distribution could be repeated, but it does not occur in all the plates. It has nothing to do with cell treatment, because it has been observed also in control cells. If you have some advice, please anwer :)
I am writing to report a concerning issue I've encountered during my recent migration assay using RAW 264.7 cells. I have followed the standard protocol, but I am facing a serious challenge with cell viability and scratch healing in response to CXCL9 treatment.
Here is a detailed description of the experiment:
Cell Line: RAW 264.7 cells
Media: DMEM Free Serum (used to standardize the results, Raw cells 264.7 grow in DMEM with 10%)
Scratch Assay Setup:
Cells were plated in 12-well plates until they reached confluence.
Media was removed, and cells were washed with PBS.
A scratch was made using a 200μl tip.
Cells were washed again with PBS.
Fresh DMEM Free Serum media containing varying doses of CXCL9 cytokines was added.
The issue I'm facing is that after the addition of CXCL9, the scratch disappears quickly and cells seem to detach and float, indicating a loss of cell viability.
I would like to inquire whether I am following the correct protocol, if a reduced FBS media is preferable over serum-free media, or if you have any alternative suggestions for conducting the migration assay with RAW cells.
If I am using the SHSY5Y cell line to test CNS regeneration, do I have to differentiate it to be more reliable?
if yes, what is the most reliable & applicable protocol?
Also, any hint on the specific CNS regeneration markers to be detected using this particular cell line?
I have developed an peptide with machine learning that targets receptors vegfr1, vegfr2, and vegfr3. I plan to test its binding specificity on each receptor and measure the downstream effects in a cell line
I tried reviving the HaCaT cell line using the standard protocol for cell revival. However, they aren't attached to the flask even after 72 hours. The cell lines were stored in August 2022 by a colleague of mine. What would be the reason behind their failure to revive?
Media used: DMEM HG 12% FBS
The recommended media by ATCC for culturing HaCaT is Dermal Cell Basal Medium (https://www.atcc.org/products/pcs-200-011#required-products). However, our lab has previously grown and cultivated them in DMEM with 12% FBS. Others have used DMEM with 5% FBS. (
Could the problem be due to the difference in media? Or the stress due to storage conditions that prevented its revival?
Are these HaCaT cells stored in -80 really lost?
Please have a look at the image.
I've cultured cells (cell lines) "stored for a long period in liquid nitrogen".
In addition to low viability, I have a problem with cell attachment to the culture flask (most cells are still in suspension), and some of them form spheroids. How to save cells that are still alive :
- Could long storage of cells cause this problem?
- Are there factors that promote cell adhesion?
- It is possible to use 3d cell culture in this case?
I used HEK293 cells as expression system, transiently transfected using PEI. Harvested on Day 4 or sometimes Day 5. The enzyme produced always show up as strong band in cell pellet in western blot , while there is no band in supernatant. While doing enzyme assay, this shows no activity.
I changed my expression system and used Expi293 cell line, which produces an active enzyme.
1. Is this related to cell lines, but in past we have seen HEK293 cell line producing any other enzymes?
2. Does vector design plays any role in this?
Hey all my cell culture fellows,
i need your help and experience...
i am trying to establish a cell line in our lab. i bought a fish cell line and i am now trying to get it startet. But it is not going as well as i hoped. The epithelial like cells are growing at 19°C with no extra CO2. I got about 1 Million cells delivered and i tried to seed them into a 25cm^2 flask, as statet in the protokoll. at first they seemed to grow fine, but in the last passage and i the passage now, the cells are distributed very unevenly in the flask. Last passage, the cells were very dense in the upper half of the flask (like if you watch the field under the mikroskop), and in the lower half, they were not as dense. And around the edges there were almost no cells growing.
In this passage, i have a small field in the middle of the flask where the cells are very dense, and around that they are not growing well. Also today on day five i have a lot of cells detached. Its very frustrating... i thought maybe the incubator is not standing right, but the distribution of the cells make no sense to me...
Has anyone any ideas?
I am currently doing qPCR to detect apoptotic gene expression in cell lines. After incubation with treatment, do i need to collect both dead and live cells OR only live cells to proceed with RNA extraction & qPCR?
Thank you in advance.
I want to study the expression of some proteins related to oxidative stress with western blot. I wonder what microglia cell line is the best for it.
I am a third year PhD student and have previously optimized a protein complemetation assay (PCA) using NanoBit technology for a high throughput drug screening. I have further validated the activity of the compounds through the assessment of downstream inflammation. However, I need to validate the PCA assay further to prove that my compounds are not interacting with the NanoLuc fragments. The initial plan was to create an NanoLuc-CyOFP HEK293T cell line and carry out the PCA with my compounds using that. However, I have been doing some research and I am worried that "components of the NanoBit optimized reporter are structurally distinct from Nanoluc" see
Will doing this further assay then not validate the compounds are not interacting with the NanoLuc fragments in the initial assay?
(Further to this, I know that a better assay would be using the same NanoBit technology with a different well known protein-complex from a different pathway, however, due to lack of time and resource I am unable to do this)
Any clarification or explanation would be appreciated.
Thank you in advance.
hello i am a new master's student in an immunology lab and I have been assigned a topic to establish a stable cell line for swine interleukin 2 gene expression, but I do not know how to start my experiment, if someone can help me in this I will be highly obliged. Thanks
Hello guy, I need your help to solve my problem in experiment. I got sample as culture cell line. I have to detect whether virus infection or not in this cell culture sample (pharmaceutics product). I should test the presence or not of RNA virus from bovine/porcine in these cell lines by quantitative real-time PCR method.
My plan is: I will find the specific sequence for these RNA viruses to design primer, probe and plasmid (that I will use as positive control when I running RT-qPCR, and make standard curve). In this case, when I perform RT-qPCR I need RNA or DNA to running.
My question here is: In this case, how can I isolate RNA or DNA of virus from culture cells? Or anyone have experience to perform this kind of experiment before can tell me what should I do in this case?
I have never do this kind of experiment before so I am so confused. Thank you so much for your help.
I am currently establishing a transgenic cell line (H9) using a plasmid containing Zeocin resistant gene. An antibiotic kill curve was performed ranging from 0.5 ug/mL to 1000 ug/mL. H9 WT cells were killed within 2 days with the lowest concentration (0.5 and 1.0 ug/mL), while all cells detached and underwent cell death within a day with other concentration. Similar rate of cell death was observed in cells containing the resistant gene where its gene expression is driven by SV40 promoter.
Therefore, I am asking if anyone has used Zeocin to maintain or select cells with Zeocin antibiotic. If yes, what is the concentration used, particularly on H9 cell lines.
The dose is standardised in animals in mg/kg.
i just wanna extend the same treatment to cell lines and was trying to find a way to convert Drug dose from mg/kg to mg/ml ?
I have worked with many cancer lines that carry various mutations and gene rearrangements. But now I have a question - are there stable (or, with a lifespan limited by the natural Hayflick limit) cell lines derived from patients with, for example, Down syndrome or Shereshevsky-Turner syndrome?
I am generating stable cell lines (HEK293A) with my gene of interest using the AAVS1 integration system. The integration site is present on Chromosome 13, which is triploid in HEK cells. I want to confirm how many copies of my gene of interest are present in the different clones I have got for my stable cell lines. One way of doing so is doing a qPCR on genomic DNA isolated from these cells but what control should I be using to estimate the copy number?
have a good time
My goal is to transfect SH-SY5Y and U87-MG cell lines using electroporation method. Has anyone had experience working with these cell lines? And what electroporation buffer should I use to transfect these?
There is a SA13 human hybridoma cell line in our lab, which - according to the rules of working with cells - is grown on a special IMDM medium. We do not have the opportunity to obtain this medium, but there are DMEM, RPMI, EMEM, alphaMEM media. Can I replace the IMDM with the ones above? Is there a critical difference?
Thanks in advance!
Is there a human neuronal cell line used as an in vitro model to apply scratch injury to perform cell migration assay?
Other than SHSY5Y as I am using it already.
If any, any special hints about its culture protocol?
I want to perform an antiviral assay for HIV inhibitors. I can't do the p-24 assay and I can only do the beta-galactosidase assay. So which cell lines can I use for this other than the tz-mbl cell?
I extracted RNA from two different types of cell lines: one that is sensitive to cisplatin and one that is resistant. I then followed the cDNA synthesis protocol and observed a strong band for the resistant cell line cDNA, but no band for the sensitive cell line. Can you help me identify the problem?
I had SH-SY-5Y cell line that was contaminated however I managed to decontaminate it using antibiotic and it worked and ended up having a good stock of the cell line with high proliferation and viability however when I tried to seed 7500 cells per well in a 96 well plate after 24 hours cells started to settle and take the morphology but once I started treatment with a drug when the media was aspired and new media with drug was added cells started to detach from the well forming batches, I tried different cell counts with different confluency and from different passages but the problem was still there
I never handle the SHSY5Y cell line before, so it is a new thing for me, as far as I observed for these past five days, their growth is really slow. Is it normal? I use the complete media of nutrient mixture F12 Ham supplemented with 15% FBS (recommendation from ECACC), I'm afraid if there might be something I missed or if there is any recommended suggestion for me. Thank you.
I have been growing SH-SY5Y cell line in DMEM/F12 media supplemented with 15% heat-inactivated FBS. For differentiation, I plated 10000 cells per well in 96 well plates and changed media every day, which contained 3% FBS with 10 micromoles RA, and also I have tried with 10% FBS and 10 micromoles RA.
In 3% FBS, I noticed cell death within two days of differentiation, and in 10% FBS after five days the well was almost filled with cells. So what should be the ideal FBS concentration and seeding density for differentiation?
I have attached image of the SH-SY5Y cell line acquired after 5 days of differentiation with 10% FBS and 10 micromoles RA most of the cells look like epithelial
I would like to assess in vitro the cytotoxic effect and possible Bruton kinase inhibition of a serries of organic compounds as potential therapeutics for the treatment of chronic lymphoblast leukemia.
Could I use K 562 cell line as a model? Could you suggest a more suitable cell model? Thank you!
I have fixed my cell (U937 cell line) using 4 % parafarmaldehyde and blocked them using 1%BSA. Can I still use TritonX100 to permealize the cell?
Hi all, I am interested in performing bulk RNA sequencing on primary human cells that have been cultured in absence/presence of certain types of drugs. I know n=6 is quoted as an acceptable number for cell lines and genetically identical mouse samples, but I can imagine the number of replicates needs to be higher when using primary human cells taken from a variety of donors. I am struggling to find any comparable published studies so I was wondering if anyone here had an idea/some experience with this? Many thanks!
I recently thawed THP-1 cells frozen at passage 20 in 2016 in 10% RPMI. After thawing, the cells appeared to recover successfully, and the next day I changed the media to remove the remaining DMSO. The cells were cultured in T-25.
On the 7thday, the two T-25 flasks showed turbid media. The other cells in the CO2 incubator with the THP-1 cells did not exhibit any additional contamination.
A microscopic examination revealed tiny critters that were moving quickly.
I suspect it might have been the frozen cell vial that was the contamination source.
Since I have been working with various cell lines, I have never seen anything like this!
Any suggestions as to what this might be, and how to get rid of it, would be of great help!
I am now trying to work with OSU-CLL cell line. Oddly, after a while of culturing (perhaps we changed the FBS), the cell started to exhibit some behavior like an adherent cell. They like to clump togehter and sink to the bottom of the culture flask, is this normal? Do you have any experience with culturing this cell line?
Thank you very much for your help！！
The cell line I used in my experiment is a self-cultured porpoise fibroblast cell line. On the first day, I seeded cells on a 96-well plate and the cell density was 5 x 10^4 cells/mL. The day after, I added drugs and went for 24hr incubation. Then, on the third day, when I was going to perform the MTS assay, I found that all the cells were dead, including the cells of the control group which were only treated with a culture medium without any added drugs. I am so puzzled and wondering why, anyone has faced the same problem and can help me.
My cell culture medium is also without FBS since I need to collect the supernatant for the DNA damage assay. The number of passages of the cell line is 19. The cells cultured in T75 flasks were growing well and a little fast than before. Some people said that it is not good if cells grow too fast, is that true? Could this be the reason for the sudden death of my cells?
Thank you so much!!!!!!
Is there anyone working with lipid-based nanoparticles (LPNs) for mRNA delivery who can share their thoughts?
Currently, I am working with PLGA:DOPE:DOTAP nanoparticles, but I am facing significant difficulties in achieving successful transfection in various cell lines (HEPG2, HEK293, JurkatE61). I have already attempted mRNA encapsulation and adsorption, but none of the formulations have yielded any transfection results.
I would greatly appreciate any insights or suggestions on improving the transfection efficiency with LPNs and mRNA delivery in these cell lines.
Thank you in advance for your assistance.
My lab has just recently got a cell line. The cells grow a bit slowly and the medium color is changing quite fast from pink to orange compared to other cell lines. In addition, we observed some strange black debris. However apparently the movement is brownian, unlike bacterial movement.
Have you ever experienced thing like this? Could you help me pinpoint what is this debris and share any suggestion what should I do? Thank you.
I have a problem with number of cells in E0771 cell line . I usually use 100-2000 cells for sphere but I couldn't get good sphere.
After analyzing PI data of LN229 cells (glioma cell line), I observed that even the control (untreated) population had the following cell cycle proportion:
G0/G1 : 79%
S : 8%
Is this a valid data? As far as cells are concerned, I don't find any morphological or divisional variation.
Hello, I am having trouble producing a protein using the Expi293 cell line.
To be clear, I have successfully produced this protein using the mother cell line but after freezing and thawing the daughter cell lines, I keep failing.
When the product first came, I thawed the cells and subcultured them until P3, which is when I froze the cells for later usage. I transfected the mother cell line with the Expifectamin transfection kit and successfully produced the proteins with good yield. The cells were subcultured until P20, which is when I discarded the cells and thawed the cyropreserved cells.
The cells divide in a healthy condition with normal morphology, but they simply fail to produce the desired proteins following transfection. I have tried using a completely freshly new transfection kit and culture media but this didn't help.
The conclusion I have reached is perhaps (by incredibly bad luck) the Expi293 cell line lost its protein producing ability during freezing procedure. Has anyone experienced such problems regarding protein production (even better if you have used the Expi293 cell line)? I would love to hear opinions and tips to make this work.
I am trying to do polysome profiling experiment with MEF cell line, but it doesn’t work ( attached the profile I got).
I did the same protocol with different cell lines ( HEKs & U87) and it works very well. I thought maybe the transcriptome in MEFs is low and I should increase my starting material ( maybe I should use two 15cm plates instead of one plate).
What do you think the problem is?and how I can improve my experiment?
I am carrying out the MTT assay using BJ-5ta fibroblasts cell line to evaluate the cytotoxicity of the nanocomposite hydrogels. I want guidance about deciding the controls in the experiment.
blank: media+MTT solution (to cancel the background noise)
positive control: Cells+media+MTT solution
control hydrogel (containing no nanoparticles)
hydrogels (containing different concentrations of the nanoparticles)
negative control : here I am confused and need guidance, whether it should be DMSO+cells+MTT solution
I will be grateful for your help!
We routinely use MTT assay to test the cytotoxicity of compounds. Recently, however, our background values of some cell lines (e.g., MCF7, MDA-MB-231) are shallow, while other cell lines give normal background levels. We excluded infection of the cultures. Have you experienced similar results? How can we fix this?
Tnaks in advance
My project includes 2 cell lines (developed from a stock culture) then each cell line was subcultured into 18 flasks. These 18 flasks are then separated into 3 temperatures and incubated for 6 time points. At each time point, I would analyze 2 flasks (one from each cell line), and this flask is discarded after.
Right now, we have the data analyzed with a mixed ANOVA model (time vs temperature as within factors) and tukey (to determine any differences between temperature, time point, and cell line). I was wondering if this is correct? Or should we do it differently because each time point uses a different flask compared to the next (meaning different cells from the stock culture).
I am trying to grow JIMT-1 (breast cancer) cell line. However, the cells fail to attach to the surface of the T25 flask. I have contacted the seller but they only provided me with a generic troubleshooting procedure, which I have been following. I want to try and recover this cell line. So, other than growth supplements, is there any suggestion to work on the same?
Hi, I want to evaluate oxidative stress in a neuroblastoma cell line, I know about SOD1, but I think it's not enough, so I was investigating and I found 4-hydroxynonenal, I have this in my lab 4-HNE B5605, but I don't find the concentration to use. Also I don't think it is the best marker for inmunofluorescence, It is more used in western blot . Do you know other types of marker that I can use for oxidative stress in a cell culture? I will appreciate your help.
How can we optimize the activity of a cell type-specific promoter driving a GFP reporter? We observed a remarkably weak expression of the reporter gene in the cell line. In contrast, native animal cells exhibit significantly higher promoter activity.
I am working with HeLa and SiHa cell lines. I would like to have one other normal cell line alongside these cancer cell lines, to identify the effect of the drug in other normal cell lines. Is it mandatory for me to take any normal cell line of cervical origin or can I use any other cell lines like fibroblast?
I want to perform MTT assay for BJ-5ta and HaCat cell lines. I want to know before performing the actual experiment how many passages should be done of the cell lines?
We are interested in transfecting a CHO cell line to produce a recombinant protein.
We are planning to use the limiting dilution method in 96-well plates to select single-cell clones to be screened for expression and we are looking for a detailed protocol for this cell line. Specifically, we would also be interest to know the cloning efficiency with this cell line (i.e. the expected ratio between wells plated and clones obtained).
I'm working with Platinum-E (Plat-E) cells and generating viral particles that carry an insert of interest with a GFP reporter that will be used to infect mouse T cells. I understand that when working with lentiviruses, you would typically infect 293T cells (or a similar cell line) and measure how many cells are positive for GFP to determine your functional viral titer. You would then move on to determine the MOI of your cell type of interest. However, retroviruses produced by Plat-E cells are ecotropic and only infect mouse/rat cells. Thus, I would have to infect a mouse cell line to determine my functional viral titer. Do researchers working with mouse retroviruses typically do this? (Determine functional viral titer by infecting mouse cells). If so, what cell line/type is typically used for determining viral titers. Or is it standard to just jump straight to infecting your cell type of interest?
Thanks in advance for any advice and information!
I have problem about MTT assay in U87 cell line. After add MTT solution in U87 cell line, the cell were floated. Who used to find the same problem. Why is cause of this action? Please answer and suggest me for solving of the problem. Thank you.
Hi I am kind of looking to see if a comparative expression level analysis of the different Glycosylation enzymes of different CHO cell lines (DG44, DXB-11 and K1)is available in any publication. I am new to this field and a little help will be greatly appreciated. Thanks in advance.
I am working on my PhD project and recently we plan to have our experiments using immortalized nasoepithelial cell line. First we tried ABM T9245 with the Prigrow serum the company suggested, with pre-coated vessels, however the cells didn't attach at all. For some other issue we asked for a compensating vial ABM T0440, along with the suggested serum and precoated culturing vessels. This is the second day after thawing, and the cells don't attach at all either.... Does anyone who worked with similar cell types have any idea what's wrong? or offer any suggestions?
I thawed the cells using 1 min 37 degree water bath, then resuspended in 5 mL Prigrow X, followed by 200g x 3 min centrifuge and finally resuspension and seeding as suggested density.
I seeded 8000 cells per well in 1 96 well plate. The number I think is very high for T84. So, I need the doubling time of T84.