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Cell Line Culture - Science method

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I am preparing to work with Caki-1 and Caki-2 cell line cultures. I've seen a couple of biotech company websites suggesting the inclusion of 2 mM Glutamine to the McCoy's Modified Media that these cells require. Some other websites have no mention of the Glutamine addition so I want to know if Glutamine is necessary. Any advice is appreciated!
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No need to supplement more. 1.5mM L-Glutamine should be sufficient.
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I recently purchased cryopreserved SW-982 cells. I thawed them according to the manufacturer’s protocol and cultured them in RPMI medium supplemented with 10% FBS, 1% antibiotics, and 1% glutamine (I’m unable to use L15 medium as my incubator is set to 5% CO2). From the start, the cells have been growing in suspension, forming spheres, with very few adhering to the flask surface.
I contacted the company, but they were unable to identify the cause of the issue.
Has anyone encountered this before? Why are they growing in this way, and what can I do to make them grow forming a monolayer?
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  • Thawing and Recovery Phase: Cells are often stressed after thawing and may take some time to fully recover and adhere. Did you change the medium 24 hours post thawing to remove any residual DMSO?
  • Confluency and Seeding Density: If the seeding density is too low, cells may struggle to adhere. Try increasing the seeding density slightly. After thawing, it’s often recommended to start with a higher seeding density to promote recovery and adherence.
  • RPMI Medium: RPMI medium should be compatible with SW-982, but you might also consider testing other media like DMEM. While L15 medium is commonly used for these cells without CO2, you could try to optimize RPMI, and adjusting the CO2 concentration might also help.
  • Cell Quality: Might be that the cells were not handled correctly during transport and maybe damaged. Ask for a replacement from the manufacturer if this is the case. Do some troubleshooting, record the observations and submit it as proof to the supplier asking for replacment.
  • Mechanical Detachment: Try gentle pipetting during media changes to see if this can break up the cell spheres and promote more even distribution and adhesion.
  • Matrix or Surface Coating: Sometimes, certain cell lines benefit from a coated surface (e.g., collagen, poly-L-lysine, or fibronectin) to help with adhesion, especially after thawing. SW-982 cells don’t typically require it, but it may help in cases of attachment issues.
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I had mammalian cancer cell line cultured and fixed for confocal imaging. While observing I found these particles around cell nuclei stained with dapi. I checked the main culture for bacteria but there was no bacterial contamination. Are these mycoplasma?
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Dear Manita,
It is possible but quite difficult to see mycoplasma with confocal imaging. You can refer to this recent article from the japan CRB Cell Bank to get an idea. This is quite cool (they discriminate contaminated cells with autofluorescence signature) ! The pictures are different from yours. Alternatively you can use our straightforward Mycostrip Kit as in the publication.
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I am currently working on iPSCs and we usually culture them in mTSER+ media. In our routine mycoplasma testing, we found that all cell lines cultured in the mTESR+ media were positive for mycoplasma, and after some investigation, identified the source was the mTESR+ 5X supplements (Stem Cell Technologies : Cat#100-0275, Lot#1000146377). Freshly thawed and opened supplements independently tested positive on two separate occasions, with two different sets of mycoplasma primers. The basal media tested negative. I was wondering if anyone else faced this issue or have product from the same lot who can verify their products as well.
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Hi there, I'm Matthew, a Product Manager for PSC Biology at STEMCELL. This looks to be the same case that was addressed in this thread on X (https://x.com/STEMCELLTech/status/1795260927324856792), in which we revealed that the test was showing false positive results for mycoplasma. If you have any further questions, please reach out to us at techsupport@stemcell.com.
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I have purchased HK-2 cell line from ATCC and using Keratinocyte serum free medium plus 0.05mg/ml BPE plus 5ng/ml EGF purchased from invitrogen. Actually, the main problem what I am facing is that after 5th day when I was ready for splitting, they become rounded off and dies suddenly. On 4th day at 4 pm i have seen that cells were fine . But entire scenario is changed at morning. The media pH and color was normal. suddenly what happened to cells that am not able to clear it. If you have any idea kindly let me know. it will b great help. Thanks
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Hello,
I have the same problem. Have you found a solution?
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Hello everyone.
I am looking for suggestions for HL60 cell line culture. Our lab is new to suspension cell culture. we bought the cell line from National Center for Cell Sciences (NCCS) Pune. Upon arrival, the cell was absolutely fine, having a doubling time of around 48 hours and we were able to freeze 10 vials with 10^6 cells/ml (90% FBS + 10% DMSO). But after 6 months in liquid Nitrogen when we started culturing from those vials it seems that the cells are not proliferating. We are seeing cell clumps forming and after 3-4 days all of these cells are dying (when checked using trypan blue). Can you help me with this problem? We are unable to point out the exact cause of the situation.
Thanks
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Hi,
You will see after 7 odd days the cells will be growing at a faster rate, until then do not split them. You can also see the colour of the media turning yellow, which is a good sign, that the media is being consumed.
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Hi everyone,
I am culturing bone marrow derived stem cell now and trying to do osteogenic differentiation. I found a lot of black dots after the cells get confluence which can be washed away with PBS, but came back again at the next day and  there are much more dots in expansion medium compared with osteogenic media.
1. These dots just vibrate at almost the same place, do not move actively. After changing media two days, the expansion media looks little yellowish and osteogenic medium seems okay.
2. We can not see the medium become cloudy and cells in flask always looks fine without any visible dots (less than 80% confluent).
3. Our expansion media is alpha-MEM + 10% FBS + 1 ng/ml FGF-b.
Can anyone tell what are these things or they are just something secreted by cells? I did not notice this phenomenon before the end of last year and it's totally the same cell and same medium. 
Cells 1-3 were in expansion medium, 4-5 were in osteogenic medium with different magnifications. 
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I wonder do you have the conclusion? thanks
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HI
i transfected adenovector to hek293 cell line. i don't know Adenovirus CPE.
this picture is hek 293 cell line 8 days post transfection. do you observe any CPE?
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Certainly, I can guide you on how to evaluate and analyze the cytopathic effect (CPE) caused by adenoviruses in cell cultures, which is crucial for identifying virus infection and determining its intensity. Adenovirus CPE involves characteristic changes in host cells induced by viral invasion, which can be used effectively to assess the progress and severity of infection. Here’s a detailed approach to study adenovirus CPE:
  1. Cell Culture Preparation:Cell Line Selection: Choose a suitable cell line that is susceptible to adenovirus infection. Human cell lines like A549 (lung carcinoma) or HEK293 (human embryonic kidney cells) are commonly used for adenovirus studies due to their high susceptibility. Culture Conditions: Maintain cells in an appropriate growth medium at 37°C with 5% CO2, ensuring they reach 70-80% confluency by the time of infection, optimal for observing CPE.
  2. Virus Inoculation:Virus Preparation: Use a known titer of adenovirus to infect the cell cultures. The amount of virus used can vary depending on the desired multiplicity of infection (MOI). Infection Process: Remove the growth medium and add the virus in a small volume of serum-free medium to allow close contact with the cells. After allowing virus adsorption for 1-2 hours, add growth medium back to the cells.
  3. Monitoring CPE:Daily Observation: Examine the cells daily using a light microscope to observe the development of CPE. Adenovirus typically induces rounding, clumping, and detachment of cells. Keep detailed records of the progression and intensity of these effects. Photographic Documentation: Capture images of the CPE at various time points post-infection to document changes and compare with uninfected control cells.
  4. Quantitative Assessment:CPE Scoring: Develop a scoring system for CPE based on severity and extent (e.g., 0 = no CPE, 1 = mild CPE, 2 = moderate CPE, 3 = severe CPE). Viability Assays: Use assays such as MTT or trypan blue exclusion to quantify cell viability and correlate with visual CPE observations.
  5. Viral Titer Estimation:Plaque Assay or TCID50: Following the observation of CPE, perform a plaque assay or TCID50 to quantify the viral titer. This helps in correlating the extent of CPE with the viral load.
  6. Data Analysis:Data Compilation: Compile observational and quantitative data to analyze the relationship between the extent of CPE, cell viability, and viral titer. Statistical Analysis: Apply appropriate statistical methods to validate the observations and assess the reproducibility of the results.
  7. Reporting and Review:Documentation: Prepare a comprehensive report detailing the methodology, observations, data analysis, and conclusions. Include all photographic evidence. Peer Review: If applicable, have the study peer-reviewed to ensure accuracy and validity of the experimental approach and findings.
This structured approach allows for a thorough investigation of adenovirus cytopathic effects, providing valuable insights into the virus-cell interactions and the efficacy of potential antiviral treatments. Regular and meticulous monitoring coupled with detailed documentation are key for a successful analysis of adenovirus CPE.
Reviewing the protocols listed here may offer further guidance in addressing this issue
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I am maintaining MCF7 in 10% FBS and 1% Penicillin/Streptomycin DMEM medium and there was a formation of aggregates after passaging by 3 days. The growth medium color wasn't changed. Could you please help me identify this contamination? The pictures were attached below.
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The bacteria were moving. We haven't known the actual source but everything is better since we have already replaced all the materials and cleaned the equipment.
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I am establishing a polarized gastric-epithelial monolayer culture on transwell system for bacterial infection studies. I use NCI-N87 cells and I culture them by replacing medium on every alternative day for 21 days. Later, I confirmed the expression of ZO-1 on 100% methanol (-20C) fixed cells. However, I face the following issues during this process.
1. How to avoid membrane curling while I cut off the membrane from the insert to mount on glass slide?
2. When I used 4% formaldehyde as a fixative to stain cell surface proteins, I found few cells or small cell clusters lying over the tight monolayer.
3. Is it necessary to use 21 days grown cells for bacterial infection studies? Because I see many highly cited papers also have used lesser days grown cells.
How to overcome these technicalities?
Any help is highly appreciated.
Thank you in advance.
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Thank you Sasikala!!
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Hi!
I am staining iNOS and CD163 in my cells so I am looking for a minus control, expressing neither iNOS nor CD163. But the cell familiar in our lab including panc-1 and 293T seems at least expressing 1 of them. Which cell line is known to not having observable level of these 2 proteins?
Thanks!
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A cell line that does not express both iNOS (inducible nitric oxide synthase) and CD163 (cluster of differentiation 163) could be a cell line derived from a tissue or cell type that does not typically express these markers under normal conditions.For example, many cancer cell lines, such as HeLa cells or MCF-7 cells, may not express iNOS or CD163 unless they are specifically induced to do so under experimental conditions. Additionally, certain immortalized cell lines derived from non-immune tissues, such as fibroblasts or epithelial cells, may not express these markers.However, it's important to note that the expression of iNOS and CD163 can vary depending on the experimental conditions and the specific context in which the cells are studied. Therefore, it's always a good idea to confirm the expression profile of a cell line using experimental techniques such as immunostaining, Western blotting, or flow cytometry.
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Has anyone had any success with Crispr and MDA-MB-231 or MCF-7 cell lines?
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Dear Colleague,
Thank you for your inquiry about the application of CRISPR/Cas9 technology in MDA-MB-231 and MCF-7 cell lines. The use of CRISPR/Cas9 for genome editing has indeed been widely adopted across various cell lines, including those relevant to breast cancer research, such as MDA-MB-231 and MCF-7. The success of CRISPR/Cas9 in these cell lines is well documented in scientific literature, underscoring its potential for understanding cancer biology and developing therapeutic strategies.
MDA-MB-231, a triple-negative breast cancer cell line, and MCF-7, an estrogen receptor-positive line, have been utilized in numerous studies to investigate the role of specific genes in cancer progression, metastasis, drug resistance, and other crucial aspects of cancer biology. CRISPR/Cas9 has been employed in these contexts to knock out genes of interest, introduce specific mutations, or modulate gene expression, thereby elucidating their functions in cancer development and progression.
Several key factors contribute to the success of CRISPR/Cas9 in these cell lines:
  1. Efficient Delivery: Achieving high efficiency of CRISPR/Cas9 delivery into MDA-MB-231 and MCF-7 cells is crucial. Techniques such as electroporation, lentiviral transduction, and lipid-mediated transfection have been successfully used. The choice of delivery method can significantly impact the editing efficiency and cell viability.
  2. Guide RNA Design: Designing highly specific guide RNAs (gRNAs) targeting the gene of interest is essential to minimize off-target effects and enhance editing specificity. Online tools and databases can aid in the design and selection of optimal gRNAs.
  3. Selection and Clonal Expansion: After introducing CRISPR/Cas9 components into the cells, selecting successfully edited cells is crucial. This often involves antibiotic selection (for plasmid vectors carrying resistance genes), followed by clonal expansion and validation of gene editing through PCR, sequencing, or functional assays.
  4. Functional Validation: Post-editing, it's imperative to validate the functional consequences of the genetic modifications. This includes assessing changes in cell phenotype, growth, migration, drug sensitivity, and other relevant cancer cell behaviors.
Literature reports demonstrate successful applications of CRISPR/Cas9 in these cell lines, ranging from basic research on gene function to more applied studies on drug resistance mechanisms and potential therapeutic targets. These studies underscore the adaptability and effectiveness of CRISPR/Cas9 in manipulating the genomes of MDA-MB-231 and MCF-7 cells for diverse research purposes.
In conclusion, CRISPR/Cas9 genome editing has been successfully applied in MDA-MB-231 and MCF-7 cell lines, contributing significantly to breast cancer research. The continued refinement of CRISPR/Cas9 methodologies and an increasing understanding of cell line-specific responses to genome editing will further enhance the utility of these cell models in cancer research.
Best regards,
Reviewing the protocols listed here may offer further guidance in addressing this issue
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I have been experiencing some trouble culturing Hep3B cells since a while now. I use DMEM with 10% FBS and PenStrep. On reviving a vial the cells grow very slow and even if they start growing well, they suddenly die and the growth is stopped altogether. 
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Have you tried with DMEM high glucose?
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I am referring here predominantly to therapeutic CHO cell lines, although this trend does seem to be widespread. In the literature, often in the same study, electroporation is used to generate stable cell lines, whereas a chemical method such as PEI-mediated transfection is utilised for transient gene expression. This is true for industry and academia as far as i can tell. Reviews on mammalian transfection methodologies tend to argue that chemical methods are by far the most common, for a list of reasons that make it more advantageous. Does anyone know of any reason why people continue with electroporation for stable work? If I was to guess I would say that it is more efficient at DNA delivery and that the hit taken in cell viability is not so important, because stable cell line generation allows plenty of time for recovery and perhaps also because regulatory bodies might not be comfortable with potential lingering chemicals in formulated products. However, I cannot find any literature to support this. Any help would be greatly appreciated. 
Thanks in advance,
Joe
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Hope someone can answer this question. From a few pieces of literature I read, electroporation could transfect more linearized plasmid into cells in a short time, which indirectly increasing the DNA concentration in nuclear. Compared to this, chemical transfection needs 4-6 hours to intake those DNA. and the transfect efficiency of linearized DNA in chemical transfection is low. Also, you cannot transfect too much DNA by using chemical transfection, which may cause some immune resistance and then decrease the transfection efficiency. For transite transfection I don't know, both electroporation and chemical transfection could be used. I guess chemical transfectin kit is much more easier to approach, and the procedure is more easier to follow.
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Two or three days after removing the differentiation medium, the cells are detached from the culturing plates. I have tried several times, ending up with the same problem. Does anyone have the same experience or have any suggestions to fix this problem?
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As I know that,
1. Make sure the confluent cell is performing at its peak before treating the cell culture with DIM by changing the medium (D+BCS) one day before. I was able to observe that the medium color changes to a yellowish hue near the confluence of the cells, which I believe accelerates the process of cell detachment (Through the use of a microscope).
2. Medium Discard: Take care to work slowly and cautiously. I used to manually use a 1000 uL micropipette and 10 microtips plus 1000 tips.
3. Using a 1000 uL tip and 1000 uL micropipette, dispense medium. Proceed cautiously and gradually from the well's wall (until covering all the cell surface), and then gradually lower (drop by drop, on the center of cell culture surface) the remaining medium.
Dispense the liquid by the well's wall, producing flow to the edge of the cell culture, in my opinion, to promote cell detachment from the edge.
thank you bi Zhang
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I cultured the Hela cell line for anticancer testing of a plant extract. On the 3rd day of subculture and changing the media, the cell culture showed that the DMEM medium was cloudy and looked like small particles when observed with an inverted microscope at 40X (as seen in the attached picture). What do you think contaminated the HeLa culture cells? Has anyone experienced the same as me?
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Whenever I come across such a situation (media turning cloudy), I doubt bacterial contamination, and without wasting much time I confirm by streaking a few microlitres of the culture on LB agar plate (already prepared and stored in the refrigerator at 4°C. They are stored upside down with the agar on the roof of the plate), and leaving the plate at 37 degrees C overnight. If bacterial colonies are found on the plate the next day, it means presence of bacterial contamination.
So, you too need to streak a few microlitres of this so-called contaminated Hela culture on LB agar plate and confirm for yourself whether the contamination is bacterial. In a similar manner check for contamination in the DMEM media as well.
Best.
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Hello all,
I have recently embarked into mammalian cell culture using mutiwell culture plates (e.g. 6-well plates, 12-well plates, etc.) as opposed to previous studies where I have used individual flasks (e.g. T25, T75, etc.). When using flasks, I was accustomed to lysing cells in somewhat of a more "low-throughput" method in a lysis buffer containing 1% Triton X-100, applying mechanical shearing force by passing the cells through an 18G needle.
Moving to multiwell culture plates, the needle method is quite tedious. Having to pass the cells from each well through an 18G needle, one at a time, is very time consuming and counterbalances much of the time that is saved by using a multiwell plate in the first place.
Is there another method that is friendlier to "high-throughput" multiwell plates, that anyone might suggest which lyses cells without having to pass them through a needle one-by-one -- yet does not interfere with downstream assays for protein concentration?
Thanks in advance,
Chris Dieni
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Dear Dr. Dieni
You may use a plate shaker with a slow speed of about 25-50rpm.
Best.
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I am working with primary adipose-derived stem cells and have the problem that after plating the cells on 12-well-plates they all move to the center of the wells. I tried several things (8-shaped shaking, different volumes and seeding densitites, incubating the cells on a flat bench for 60 min before putting them into the incubator etc.) which all did not solve the problem. Plating other cells (for example NIH-3T3 cells does not lead to the problem - the distribution is very even so that I assume that it's a cell type-specific problem). Now my hypothesis is that the vibrations of the used incubator is the main issue. Are there any possibilities to somehow catch the vibrations inside the incubator? I found some "anti vibrating pads" in the internet for example. Maybe someone has another idea.
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Does anyone is culturing the Wehi-3 cell line? We got it from ATCC and following the ATCC guidelines but cells are not dividing/ numbers not increasing.
Can anyone suggest
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Thanks. I will try
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Do I need to add osteogenic induction media to hFOB1.19 (CRL-11372) cell line to hFOB1.19 (CRL-11372) cell line if I want the cells to differentiate to mature osteoblast? or these cells are osteoblast already? This is my first time hOB cell line culture. Some studies incubate at temperature 39.5°C for differentiating temperature. Is it necessary? or is it OK with Growth Conditions at temp of 34°C? ( recommended by manufacturer )
- If osteogenic media need to be added the media would contains Ham's F12 Medium Dulbecco's Modified Eagle's Medium, with 2.5 mM L-glutamine (without phenol red), 0.3 mg/ml G418, 10% fetal bovine serum supplemented with 0.1 µM dexamethasone, 50 μM ascorbic acid and 10 mM β-glycerol phosphate
Thank you
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lse du Preez1,2*, Wim Richter1, Dirk van Papendorp2, Annie hFOB 1.19 osteoblast cells grown on a biomimetic biphasic nanoscaffold: anin vitro evaluation for possible bone tissue engineeringBiomedical Research 2018; 29 (11): 2442-2448
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Hello,
I have been trying to passage ma-mel-66a cell line, however, I am having zero luck. I have used RPMI + 10%FBS and RPMI + 5%FBS, I even plated them (straight from LN2) in a small p10 to help them out. Nope, nodda, nothing. Can anyone recommend me a useful protocol or media formulation for this cell line?
Thanks!
J
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There are a few things you can try to improve your passage success with the MA-mel-66a cell line:
  1. Check the viability of your cells before plating them. This can be done using a trypan blue exclusion test or a similar method. If the viability is low, this could be the reason for the lack of growth.
  2. Try using a different type of media. Different cell lines have different growth requirements and some may not grow well in RPMI + 10% FBS or RPMI + 5% FBS. You can try using a media specifically formulated for melanoma cells, such as DMEM/F12 with 1x NCSU-23 supplement and 5% FBS.
  3. Consider changing your plating density. If you are plating at a high density, the cells may not have enough room to grow and divide, which could be impacting their ability to form colonies. Try plating at a lower density, such as 1-2 x 10^4 cells/cm^2, and see if that improves growth.
  4. Check the temperature and humidity of your incubator. Some cells lines require a specific temperature and humidity range to grow well. MA-mel-66a cells should be grown at 37 °C and 5% CO2.
  5. Confirm the cell line's authenticity by using a cell line authentication assay such as Short Tandem Repeat (STR) analysis. It's also worth to try to get a sample from a different supplier.
  6. If you still face difficulty, it might be helpful to consult a lab that has experience with this cell line to get more specific advice.
I hope this helps and good luck with your cell culture!
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Dear all,
I am starting working with E0771 cell line since I have to establish an orthotopic breast tumor model in c57 mice but I have no experience with this cell line so I would really appreciate any advice that you can give me.
In particular, I saw the ATCC website and they say that it is better to culture these cells in a t-75 corning flask, maintaining cultures at a cell concentration between 6 x 10^4 and 8 x 10^4 cells/cm2, is this true also for your experience? How many cells do you plate in a 75 cm2 flask?
Do they grow fast? How many times per week do you subculture them?
Sorry for all of these questions but I am new with these cells and so I would really be very grateful for all your advice,
Thanks a lot,
Giulia
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Hi,
It doesn't matter what you culture the cells in, I culture mine in 10cm plates and they are perfectly happy. They do grow quite fast, if you use them regularly, splitting them 1:10 usually means splitting them 2-3 times a week. They are fine with being split more harshly if you don't need them as much and want to reduce the number of splits. Be careful that you don't leave them confluent for too long, as with most cells, they're not happy like that, especially since they use the nutrients in the media quite quickly.
Good luck with them!
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I have experienced this problem previously with a commercial cell line, however in this case, a well established immortalized cell line when cultured from frozen stock has cell populations that have a bright border around them. This has never been the issue with this particular immortalized cell line. I am wondering if there could be possible mycoplasma contamination. Any comments on this are highly appreciated. Thanks!
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This phenomena has nothing to do with infection, nor with contamination!
The phenomena you are describing is called birefringence. It results from the curvature of the cell membrane diffracting light. As the light coverages at your focal plain, it reinforces and this appears brighter.
You haven't seen this before? When was the last time you re-aligned the optics of your microscope? I suspect that your phase contrast rings are misaligned.
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Hi!
I need to prove that my protein is localized on the cell-surface but not inside the cell in soluble form.
How can I make it?
There is an opinion to wash cells, to label all cell-surface proteins with NHS-biotin, and to collect labelled fraction of cell-surface proteins with streptavidin magnetic beads. After that I can make western blot with specific Ab.
Maybe there is another way?
Protein and cell lines are human.
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Chad M. Petit Can you share protocol to detect cell surface protein without permeabilization?
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Wondering if TrypLE Express cell dissociation agent is significantly better than other options for culturing mammalian cell lines. Currently mainly using Accutase (for fibroblasts and epithelial cells, NOT stem cells) and sometimes also trypsin for specific cell lines. Any positive/negative experience with TrypLE Express enzyme? Main attraction is that it does not require neutralization as trypsin does, but has longer incubation times. Would appreciate any comments and suggestions.
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I grow primary lung epithelial cells and use TryplE to lift the cells.
I don't really have a problem with the "long" incubation time (I use 5 minutes), however sometimes when my cells are particularly well adhered I need three incubations (this doesn't seem to affect viability particularly).
In saying that, I've not used accutase much, so I can' really comment on the comparison of the two.
Best of luck!
Sam
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Does anyone have experience with CAEV in immortalized cell lines? Some publications show use of goat synovial membrane cells or goat milk epithelial cells, but these cell lines are not available through commercial companies.
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we are using primay cells from epidimymis making immortalization. it looks promising.
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The Boyden chamber protocol requires a high budget for us. We want to try modifying this test instead. Can we combine it with a protocol like the filter diffusion protocol?
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First of all, thank you for your reply. I'm going to do research on that. It took me so long to respond because of some health issues. Thanks for helping. Yuning Hou
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I am currently culturing NK92 cell line in complete RPMI supplemented with 500u/ml IL-2. Observing steady-state cells by fluorescence microscopy, I observed that the majority of them have a rounded but sometimes irregular shape, with a single nucleous showing lobes. However, about 10% of them showed a more regular rounded shape and a perfectly spherical nucleus.
Since this morphology correlated with the expression of a protein of interest, I was wandering if someone could tell me if the "more regular" shape could correlate to any functional state for these cells. 
thanks!
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Hi , I think it's a good idea , recently I culture NK92 cell line , and find the same situation , I advance you can extract some protein such as NK activiated or inhibitory receptors , compare them maybe find some interested.
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Hello everyone,
According to the analysis of cell-surface markers using flow cytometry, is it possible that trypsinization would block or even digest the markers?
If so, based on your experience, what is the best way to dissociate the adherent cells from the flask? Thanks
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The quality of analysis in terms of detection of surface markers using flow cytometry will be determined by the cell detachment method.
The commonly used methods of adherent cell detachment include enzymatic and mechanical treatments. Enzymatic detachment, however, can lead to significant changes in cell membrane protein structure and composition leading potentially to significant experimental bias.
Accutase which is an enzyme mixture with proteolytic and collagenolytic activities is usually considered a less damaging agent than trypsin and is recommended for the treatment of sensitive cells as well as for analysis of surface markers using flow cytometry.
On the other hand, detachment by the mechanical method is achieved using the so-called ‘rubber policeman’ which is a rubber or plastic scraper attached to a glass rod. But again, mechanical methods can lead to cell membrane damage and to substantial changes in its structure.
Thus, choosing an inappropriate detachment method may cause unintended effects biasing experiment results. So, I would suggest that you conduct a pilot experiment to assess the optimal cell harvesting method (either accutase treatment or mechanical detachment using a cell scrapper) for your cells to avoid experimental bias and obtain reliable results.
Best.
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Hi Everyone,
I am culturing the primary cells (Monocytes) from the patient blood samples. During the culture (2-3 weeks), I have seen the long tape shape black color filament (1 or 2 in number). Is it a type of contamination? How to overcome this?
Thank you in advanced.
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Doesn't seem like a contamination, but more of a poly(propylene)fiber filament, a result from plastic injection of pipette tip during production. We usually ignore it, through centrifugation it will disappear, eventually. Else keep an eye on the tip, if one showed with filament seems going to detach, please discard it and choose a new one.
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Hi Everyone,
Should I add L-glutamine during media preparation if the culture media (RPMI 1640 with l-glutamine) already contain the same?
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I aslo think that checking the purchaser information for cells/media would help. And, addition of L-glutamine to medium already containing l-glutamine is mostly not necessary.
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Hello,
I am working with M93-047 cells and I have cell lines cultured in T75 Flasks, I want to seed cell for Pull down IP experiments. I want to seed cells either in 3 or 4 10cm plates, so that I can have 3 or 4 samples of the cell line to work on.
I am confused that what dilution of RPMI media I should use to seed M93 cells, so if someone can help, and then secondly after counting the viable cells using hemocytometer, what factors we should keep in mind for seeding.
If someone can share good protocol for seeding cells, I'll appreciate.
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Thank you so much for answering my question, it’s a big help.
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Hi everyone,
I have seen some very strange debris in my U87MG (glioblastoma cell line) culture. It seems that there are shards/fragments of glass that are not contaminating the cell culture and the cells are potentially trying to adhere to them. I attached some pictures of the debris, taken shortly after passaging, so they are not fully adhered.
When I first saw this, I bleached the cells, threw out the package of flasks I was using, made new media, etc. I figured that this originated from a glass pasteur pipette, so I thawed another vial of cells that was frozen down at a different time than the previous ones I used. However, I'm still seeing the same debris in the freshly woken up cells.
I'm confused because I didn't see the debris in the first set of cells until weeks into using them, and I didn't see them in the second set of cells until about two weeks since I thawed them. I may have just not seen it until two weeks in, but it just seems unlikely due to how much and how obvious it is.
This is the only cell line I use EMEM (w/ penstrep & 10% FBS) for, and I have never seen this in any of my other cell types that use other media (completed with the same batch of penstrep and FBS).
Please let me know if you have ever seen anything like this before! Any comments are appreciated - thank you all in advance.
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Hello Nicole,
I sometimes saw them after defrosting the cells. Such structures are formed by DMSO if it is unevenly mixed when cells are frozen.
When adding to the freezing medium, try to stir the DMSO as vigorously as possible. You can also add DMSO directly to the cryovials containing media and cells and mix thoroughly. A small volume of DMSO is easier to mix.
Good luck!
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What is the time taken to reach confluence for hepg2, huh7, heap 16 cell lines cultured in 10%FBS, DMEM high glucose
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I am trying to induce inflammation in the cell lines (Beas 2B & NHBE) by using the dust but haven't worked with cell lines previously. Kindly help with how to identify the cell lines whether it is inflammated or not?
What are all the parameters I have to consider?
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Hi Deepdarshan.
BEAS-2B is a human lung epithelial cell line that shows prominent inflammatory responses after induction with Dust. As I am working on Fine particulate matter induced lung inflammation So I would suggest you to plate 8-10k cells per/well in a 96 well plate and check for the cell viability with varying doses of dust particles treatment. Further you can choose the most significant dose where cell viability is reduced and there is an increased expression of inflammatory proteins and an decreased expression of anti-oxidant proteins (can be done by immunoblotting of immunofluorescence). You can also measure the ROS by CM-H2DCFDA or MitoSOX to conclude the oxidative effect of dust particles in lung epithelial cells.
Thanks
Hope it helps...!!
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Is it ok to grow GRANTA-519, with RPMI-1640+10 %FBS?. When I tried then found that these cell lines are also growing within this media.
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How does the passage number of a cell line affect experiment results including toxicity assays? Which characteristics of cells are changing as the passage number increases?
What is the most efficient or optimum passage number of cells (for example, for cancer cell lines such as HepG2, A549 etc. or for healthy cell lines like HEK293) for setting an experiment?
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The cell will drift over passages. Usually, I would use the cells within a 5 passage range for one study. Doing cell authentication before and after the study is recommended and sometimes required. I rarely use cells above P20 for any cell line. The longer your passage an immortal cell line, you are selecting for the faster growing population of cells. Prepare a low passage master cell bank and a large working cell bank for each cell line. For each experiment, thaw a vial from the working cell bank and replace cells after 5 passages from thawing. This way, you will have a more consistent result.
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I've been trying to differentiating SH-SY5Y cells with brain-derived neurotrophic factor (BDNF) in 8-well chamber slides but I seem to be having problems with cells detaching over the course of the 7 days in serum-free media with BDNF. The problem seems to be more apparent after changing the media but I am unsure as to why this would occur and would value any insight from people with similar problems with SH-SY5Y or other cell types. My first thought was that I was removing too much media from the well, so I began to leave around 1/4 of the old media and add 3/4 new media and pipette the media off very slowly and add the new media very slowly (to the side of the well, not directly on to the top of the cells) but I still seem to be having the same issue. I thought there might be a problem with the incubator I was using so I changed incubators, and yet the problem persists.
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Did you coat with PLL?
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I thawed a tube of Calu-3 cells from ATCC, following product instructions. The flask I used was not coated with any substrate. I used EMEM with 10% FBS and placed it in the usual 37degC incubator with 5% CO2 and water pan. The next day I discarded floating cells. Since then, these cells have always appeared round and bright (see attached photomicrograph). They appear to be able to change positions, moving from middle of vessel to the sides. At first the numbers grew slowly but later it started dying. I have changed the media to DMEM with sodium pyruvate and 10% FBS. I had transferred floating cells to another vessel coated with collagen IV. I had also trypsinized one flask and transferred all cells to one collagen IV-coated well of a 12-well plate, and refreshed media every 3 days. Each day I waited for it to adhere firmly and resemble the clumps of cells I saw in the ATCC photomicrographs. But after a month, they are still round and bright. Now, they are starting to die off. Can anyone advice or suggest what I can do to rescue these cells?
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We coated our flask/plate with 10ug/mL collagen IV. Is this concentration too low compared to your protocol for substrate coating to support growth/adherence?
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I recently sequenced (Sanger) an exon from single alleles of a CRISPR-Cas9 mutated cell line (diploid) to find whether the mutation is homozygous or heterozygous and what the mutation is. What I got is 3 different results, a WT, and two diff mutations, a 1 base del and a 7 base del. My lab has come to the conclusion that it must somehow have 3 alleles instead of 2, and suggested looking into this. I can't really find anything relevant to this find, so I'm wondering if anyone might know of such research articles or point me in the right direction, and if this is even possible?
My original thought was that perhaps the 7 base deletion is somehow an error due to the crispr process? But more experienced people than me have suggested it means there are 3 alleles, although like I said I'm struggling to find relevant literature to support this.
Any help would be much appreciated, thanks.
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My thought is that you have a mixed population of cells, each with their own alleles. Streak/dilute them out to single cell colonies and screen them again.
No, your cells are not magically triploid.
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I have difficulty every time whenever I subculture C6/36 Cell line and vero cell line.
Can anyone suggest how to spread cells evenly in growth media. 
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How many Vero cells are in one T75 flask
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Kavitha Jade Brunner it sounds to me like you're doing the right things regarding the water in the water baths. As for cleaning surfaces, using the bleach wouldn't cause contamination, it can just wear down on the surfaces over time.
Disinfecting the scope and surrounding areas as best you can will likely help quite a bit! I'd love to hear if it helps as I am quite curious as to your contamination source as well, now.
I'm not sure why you don't filter media, but perhaps there is something about these cells/this media that I'm unfamiliar with, which is certainly a possibility! Since you're using antibiotics, it can be good to regularly test for mycoplasma as well if you don't already. Though you may not have visible contamination in all your flasks, contamination can sometimes be masked by antibiotics. It seems to be a point of contention among scientists (much like "a-POP-tosis" versus "a-PUH-tosis" haha) but I personally prefer to culture cells without antibiotics. If your aseptic technique is good (sounds like yours is - your source of contamination is likely from the dissection conditions), there's really no need for antibiotics in my opinion! Your PI may vehemently disagree, but that's just my two-cents. Good luck and feel free to keep me updated as to whether the scope disinfection helps!
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In my experiments, I found that different monoclonal cell lines from the same host cell produce similar concentrations of ammonium ion when using the same medium; but when the same monoclonal cell line cultured with different types of fermentation medium, the concentrations of ammonium ion vary greatly. Is it because which of the ingredients in the medium is different that makes this difference?
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Hello Xukun Xu
The components in the medium affect the concentration of ammonium ions. Thus, to minimize both lactic acid and ammonium production, glucose as well as glutamine may need to be simultaneously controlled or replaced. You may refer to the article attached for more information on this subject.
Best.
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I just got a new stock of NR-6 cell line (Mouse embryo fibroblast cells that don't express EGFR) and I need more information about the culturing and specifications of it.
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The above described the culture media. The best is to ask the company or lab you got the cells. They are the ones who culture and make the stock. Alternatively, you can search using PubMed or another. Good luck.
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I want to know the expression change of my target protein by western blot from the control and drug-treated cells. 48hrs. after the treatment, I have pelleted the cells and extracted the proteins using RIPA buffer (50mM Tris-HCl pH 7.6, 150mM NaCl, 2% NP-40, 1% SDC, 0.1% SDS, 2mM EDTA). After denatured the proteins using SDS-PAGE loading buffer, I have loaded the samples in SDS-PAGE wells. But, some of the samples leaked from the resolving gel and it did not stack properly. The samples spread laterally as soon as they entered resolving gel. I have prepared fresh RIPA also, but the result did not improve. As a control, I have run my old extract in the same gel. but it did not leak. My labmates use the same reagent, but they do not face any problems. Previously I have thought that it may be due to sample overloading so I have reduced the volume. But it did not seem the actual cause. It would be very helpful if anyone inform me of the root cause of this type of sample spreading in the gel. Please find the images, attached below.
Thank you
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SDS-PAGE gels have a limited shelf life, in an air-tight container wrapped in wet tissues about 2 weeks. Disassemble a casette to check that the gel is firm and well formed.
Also, I'd reduce the salt concentration in the homogenisation buffer to, say, 50 mM.
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Hi everyone, I am doing 2D monolayer cell culture on HeLa cells and A549 cells purchased from ATCC. I have had some problems after a few passages that cells occasionally all become apoptotic-ish looking. The cell confluence is normally high in that case, and instead of a more elongated morphology like what they normally look like, the cells can become round and flat (I cannot distinguish the contrast between nucleus and cytoplasm using bright field anymore) within one or two days. I have attached a picture of the HeLa cells that went wrong/abnormal looking below.
The media I am currently using is DMEM Phenol red free.
Can someone please provide some suggestion on this wether it could be caused by infection, medium related issue, or related to sub-culture procedure.
Thank you very much!
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Some suggestions:
1. May be passage them when they are 60-70% confluent?
2. Check the passage number? I know they say you can keep these cells growing forever but you could try a different batch?
3. Remove the DMSO completely when you plate them from stock. I used to thaw cells by adding 1 ml warm media drop by drop while holding quite tightly in my hand and then mix them to get a single cell suspension. Next, add them to warm media, centrifuge at 200 g for 5 mins, then plate the pellet in fresh media and off yoy go. Residual DMSO can cause some damage to freshly thawed cells (unless you change the media within 18-24 hours)
4. I use RPMI media, 10% FBS, l-glut, gentamycin.
5. Check whether the media is high or low glucose and see which works best.
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I am using G418 to select for stably transduced 22Rv1-luciferase expressing cells. Would anyone know of the minimal effective concentration of G418 required to kill 22Rv1 parental cells? I am currently using 1 mg/ml in my media, but I'm not seeing much cell death.
Thank you!
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Yes it is very inefficient for some cell types. Requires very high concentration for some cells.
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I'm working with BJAB lymphocytic cell line, culturing them with RPMI media (with L-glutamine) + 10%FBS + 1% antibiotics. After two days of changing media by centrifugation, they started to adhere to the T flask and a monolayer has formed. Does someone know what could be happening? Is the media lacking something or should it be a step in the handling that I'm missing?
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Sandra Cortes you should check the type of culture flask first that should not be tissue-culture treated one, and maintain cell culture with requires agitation (i.e., shaking or stirring) for adequate gas exchange. Try this maybe this can help you.
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I'm focusing on L929 cells to collect the supernatant using for Bone-marrow-derived macrophage (BMDM) cell culture.
Our L929-Conditioned Medium was out of stock and I started thawing an old L929 stock (in -80°C fridge) (it is written 2014 but maybe even before that time).
I followed the protocol of our lab to grow the cells and collect the supernatant.
Our lab used that protocol for very long time and it still worked well.
However, now the BMDM cells which I cultured by using that supernatant grew not as well as normal, the morphology changed.
Then, one of my senior who has been working long with L929 cells said that my L929 cells after 100% confluent continue growing too strongly, which forms several layers of cells.
She said it was abnormal because based on her experience, after 100% confluent, L929 cells will be inhibit somehow to hardly multiple as many as that.
(The L929 supernatant stock I used before was from her and It did work well, now I started thawing new one. )
That might be the reason why my L929-conditioned medium was so poor in nutrition, and might be also the reason why my BMDM growth medium didn't work well as normal.
We think it is because of the degeneration of L929 cells.
Does anyone have any experiences with this? Please let me know. Thank you so much.
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dear Anh
regarding the L929 cells I prepared it so many times during my PhD and it was working very well. I usually use Triple layer flasks to have a good amount of the supernatant. In your case you can do a flow cyometry for macrophage markers F4/80 and CD11b in order to convince that all your cells are macropages not other cells. I would also recommend using less passage for L929 cells no more than P5.
Kind regards
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I am trying to culture dental pulp stem cell line from our storage. The cell line was preserved on 2019. However, the cells aren't adhere to the flask. I used DMEM with 20% FBS, 1% penicillin and streptomycin, 10mM L.Ascorbic, 1mM L.Glutamin. I tried different cryovial from our storage but none of them tend to adhere to the flask.
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It seems that there is some issue either with the cell line or the culture flask itself. Did you check the viability of non-adharent cells in the flask after 24 hr of seeding? Did you find any cell adhared to the flask? If no, kindly get one vial from other sources to rule out any issue related to the cell line storage/damage. You can also check the final pH of the culture medium. With this, hope you may get the issue resolved. Good luck
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Gentlemen,
We are preparing a project to access a grant, but we are missing a lab that can help in the first phase of Proof of Concept and Safety.
The lab should be European (But not spanish, and sorry, but not UK) and be seasoned in brain samples handling, and also if possible having experience in cell morphology study (Neuron, Microglia, Astrocytes) or metabolic study (on said samples)
The project includes the development of a protocol in humans but we need to try it before on brain samples, and we are in a hurry to find a lab that can provide experience in this field.
Since we are in a hurry, you might contact me directly or respond in the thread.
Sincerely, J Vigil
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Please have look on our(Eminent Biosciences (EMBS)) collaborations.. and let me know if interested to associate with us
Our recent publications In collaborations with industries and academia in India and world wide.
EMBS publication In association with Universidad Tecnológica Metropolitana, Santiago, Chile. Publication Link: https://pubmed.ncbi.nlm.nih.gov/33397265/
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EMBS publication In association with Icahn Institute of Genomics and Multiscale Biology,, Mount Sinai Health System, Manhattan, NY, USA. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/29199918
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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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Suppose i got a cell line say for example (CHO) and i want to maintain it for my experiments, I'ld like to know what all the properties/characteristics I've to know about the cell line, I'm thinking few points like :
Doubling time, It's morphology, To know whether it is adherent/suspension cell line, It's Suitable media.
I'ld like to know what all other things I should know ?
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You will receive a product sheet with all of the cell lines you have purchased, which contains detailed information on how to handle the cells. The product sheet also includes batch-specific information like the number of cells per vial, the recommended split or subcultivation ratio, and, if available, the passage number. Prepare the appropriate medium, serum, and other reagents for reviving cell lines by assembling the necessary growth medium, serum, and other reagents. Many of these items are available from suppliers and can be combined with cell lines to create a custom order.
After receiving your desired cell line, whether adherent or suspension, thaw the cell as directed on the product sheet, count the cells using a hemocytometer, and reseeded into a culture flask based on the cell count. Finally, examine the culture flask under an inverted microscope the day after it has been thawed to look for any anomalies.
A new cell line is being studied. Even if it has been done before, it is better to repeat the procedure by performing a simple karyotyping test because there is a chance that some important characteristic of the cell has been lost due to prolonged passaging.
If the cell characterization has been passed, then propagate your desired cell line and prepare for the stock by using the recommended freezing media according to the product sheet of the supplier and transferring the frozen cell line into the liquid nitrogen vapor phase as early as possible.
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I want to add rat tail collagen (type 1) to the DMEM medium for an in vitro animal cell culture experiment. Suggest me an alternative for dissolving collagen other than acetic acid.
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I am passaging HEK 293 cells for retroviral transfection.
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Passaging is required when cells have proliferated to confluences around 80%
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I am doing a cell culturing experiment in a fluid flow chamber made of PDMS material. I was wondering how to sterilize the pipes and the flow chamber before/after the experiment, in case I want to use the same chamber for the second and third biological repeats?
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Hi,
We used devices repeatedly made up of PDMS as well as other materials with pathogenic bacteria. As long as the materials are Autoclaveable there is no problem in reusing them. Infact the current scenario of having sustainable development contains inherent clause of showing how we can use the same devices for longer periods with use of minimal processing. Apart form that, technically PDMS has shown to provide good results as long as the devices made from it are sterilized meticulously. It all percolates down to how good your skills are while handling cell cultures!!!
Regards
Amit
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Hi, I had a lot of problems with this cell line. When I count the number of cells, I can't do it, because the cells form a lot of cell aggregations and i can't separate them. The cells was incubated with trypsin for 2 minutes, and the cells were detached of plate, but the cells form aggregations. So, i have a lot of problems with the MTT assay due to the number of cells is not very real. If somebody can help me... I dont' know if the problem could be the medium, because i was used DMEM instead of McCoy...and i don't know if could be this the problem.
Thanks!!
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THANK YOU Ajay Kumar, for suggesting DMEM media for HCT116 cell line, but there are many types of DMEM media, which kind do you use to easily grow the cells? can you please share the catalog number of DMEM media of Himedia company?
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Using the recommended media and temperature to grow the cells but they die following the first sub-culture. When revived from frozen they look very healthy but as soon as they are split most of the cells die. Have tried different batches of FBS to no avail. Does anyone have any ideas?
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Lindsay M Maclellan I solved my problem with hFOB 1.19 culturing them in ploly-L-Lys pre-treated plates. And the cells grows so fast at 37°, even reach almost 100% of confluency in 5 days.
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Hi everyone,
I have been culturing MC38 cells for a few months and I am experiencing some problems. The cells aggregate a lot and generate clumps on the flask surface. I am using Trypsin to split them but it is really hard to de-attach them once they have created these clumps. I started splitting them 1/10 every three days and then switch to 1/20 to see if it improved. The viability tends to be low as well, around 50-70%. I am using DMEM+10%FBS as culture medium. If anyone has any suggestions and recommendations on how to culture these cells it would be really helpful. Thank you
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To break the clumps into single cell you can use vigorous pepetting.
Wash the cells twice with 1x PBS before trypsnization.
The moment your cell became single while seeding, you will get rid of this clumping problem.
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I am currently culturing some HEK cells with absolutely no issue, they are being cultured with the standard DMEM + 10% FBS media.
For a particular experiment, I need to introduce exogenous insulin for a variety of different time points and I am noticing that my cells are dying within 5-10 min of insulin addition.
I am adding insulin in at a concentration of 10ug/ml. The cells are grown in a 6 well and all those that dont have insulin added to them are surviving but the addition of insulin is resulting in all the cells lifting within 5 minutes. If someone could provide some guidance.
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Dear Smarth!
Plese You look a t the article
Perhaps insulin through its receptors on HEK293 cells enhances the accumulation of APP, which is toxic to these cells
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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,
Recently I thawed couple of cell lines, RPMI8226 and JJN3 and they both didn't do well during my first passage.
I thawed the vials in 37 deg water bath for couple of minutes. Transferred the cells with media into a conical tube. Centrifuged the tube at the lowest speed for 5mins. Aspyrated the supernatant and resuspended the cells in 10mL media in T-75 flask for 3 days.
I use the following media:
Gibco RPMI 1640 with L-glutamine + Penstrep + Glutamine + 10% FBS
My percent live cells was very low when I checked them 3 days later. I am thinking of using 20% FBS next time I thaw cells. Is there anything else I can do differently to keep the cells alive?
Thank you!
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Hello,
As Joseph mentioned, you have to be careful for how long you´re warming the cells, thawing freezed cells has to be a fast process, since DMSO is toxic for cells and can be the reason why you have low percent of live cells. Remember to check for any thawing specifications regarding your cell lines with your supplier.
Basically, both Freshney´s Culture of Animal Cells textbook and Thermo Fisher Scientific´s webpage recommend the following general thawing protocol:
1) Remove the cryovial from liquid nitrogen or -80°C freezer and immediately place it in a 37°C water bath. 2) Swirl the cells until you see a small bit of ice in the cryovial (this shall take about one minute). 3) Transfer the cryovial to a laminar flow-hood, open it, and transfer the thawed cells to a centrifuge tube. 4) Add pre-warmed medium appropriate for your cell line in a dropwise matter. 5) Centrifuge the cell suspension at approximately 200 g for 5-10 minutes (this vary with your cell line, I recommend you to check). 6) Decant the supernatant, resuspend the cells with medium, and transfer to a culture flask.
Note: some cells can be thawed without the centrifugation step, you can dilute the cryoprotectant with fresh pre-warmed medium, but you must change the medium the day after thawing.
Suggestions: make sure to work fast but aware of the steps of the process; while thawing the cells in the water bath make sure not to submerge the cryovial since this can increase the probability of contamination; try not to thaw cells that were freezed a long time ago, since viability can decrease; remember to always work aseptically; double check the label of the cryovial to make sure of the identity of the cells; plate thawed cells at high density to increase recovery.
In case you want to read more about thawing, Freshney´s textobook is very useful: Freshney, R. I. (2006). Basic principles of cell culture. Culture of cells for tissue engineering, 3-22.; you can also visit Thermo Fisher Scientific website, which has a video and a guideline regarding the thawing process: https://www.thermofisher.com/cr/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/thawing-cells.html
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I am culturing NK3.3 cell line. I do not have any experience with them. Unfortuately, they do not grow and I do not know why.
I am using AdvancedMEM media, 10%FBS, 1%P/S, 1% glutamine and I add IL-2 separately.(I aliquoted IL-2, so I heat my media in water bath, také out how much I need and add IL-2 directly there to avoid degradation of IL-2 while it is in the water bath. I dont know if this is the best solution?). I use100U/mL of IL-2.
When I count my cells, it has never been more than 250 000 NKs there (its been three weeks already, I should have more!!). When passaging, I leave 600mL of old media and add 2mL of new media with IL-2. I put the cells so that T-25 flask is "standing" with the cap up.
I passage them 2x per week. Whenever I count them there are quite a lot of dead cells visible.
Any idea on what am I doing wrong? Any suggestions/advice would be amazing.
Thank you so much!
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Dear Hana!
Mode of Growth:
  • Cells grow in suspension. Feed cells every 3-4 days. Cells are maintained in T25 or T75 flasks kept upright in the incubator with 6% CO2. The upright position favors cell-cell interaction, which appears to help them grow.
Method of Passage:
  • Draw off the half to 2/3's of old medium without disturbing the cells at the bottom of the flask. Resuspend cells in the rest and count. Resuspend cells in fresh, warm medium at a concentration of 3 x 105 cells per ml. Do not have a total volume exceeding 20 ml in a T25 or 50 ml in a T75 flask.
Culture Medium:
  • RPMI 1640 (pH 7.2)
  • 15% heat inactivated fetal bovine serum (FBS)
  • 15% PHA-stimulated PBL supernatant as IL-2 source (e.g. Lymphocult-T from Biotest, No. 811010)
  • 2 mM L-glutamine
  • 10-25 mM HEPES
  • 2 g/l Sodium Bicarbonate (NaHCO3)
  • 50-100 IU/ml Penicillin,  50-100 µg/ml Streptomycin
  • Try this protocol.
  • You said that you put the bottles vertically with the lid up, usually the bottles are placed horizontally in the incubator so that the surface area that is washed by the medium is larger, then there will be more cells. Even when transplanting cells, it is necessary to prepare a new medium with serum and IL-2 each time, and not to take the used medium and add 2 ml of new medium to the tube. In the used (conditioned medium), the cells metabolism cells accumulate, they inhibit cell growth.
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I searched a few papers but nowhere could find the answer. Asking out of curiosity. I went back to the first few papers that talked about RAW 264, RAW 309 cells etc. also I checked on the official webpage of Abelson's Lab from NCI, NIH. I couldn't find the answer. 
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Named after the three authors on the original paper
Ralph, rAschke, Watson
  • DOI:10.1016/0092-8674(78)90101-0
Corpus ID: 5529220
Functional macrophage cell lines transformed by abelson leukemia virus
  • W. Raschke, S. Baird, +1 author I. Nakoinz
  • Published 1978
  • Biology, Medicine
  • Cell
Abstract Three cloned cell lines have been established from murine tumors induced with Abelson leukemia virus which express properties of macrophages. Two of the three original tumors in addition yielded lymphocyte cell lines, one typical of the Abelson virus disease and the other a thymic lymphoma. Two of the macrophage lines are tumorigenic when placed in syngeneic mice. All of the macrophage lines pinocytose neutral red, phagocytose zymosan and latex beads, mediate antibody-dependent killing…
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I am working with raw cell line from last one year and they were fine before .But since last month they keep on self activating.. ( without treating any activating agent like LPS) even after thawing during 1st passage their morphology changes completely from round to star shape. 
I have tried using different stocks...
All other cell lines in our incubator are fine so I think its not incubator problem, even then i tried to clean it all over again but all in vain..
what should I try .. ??
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RAW 264.7 cells will self-activate when grown to high density before splitting. Keeping them at lower density should stop the self-activation.
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I had a contaminated cell culture I suspected several things, including the media. I put the media (only) in a T-25 flask and used 2 agar plates to check bacterial contamination (one opened in the laminar air flow the other in the CO2 incubator) and left them is in the CO2 incubator at 37 oC (after I cleaned it),
two days later I didn't find any thing on the agar plates and no changes in color or clarity of the media in flask however I examined the flask under the microscope and found these things .. What could it be?
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I understand that this can be very frustrating. The other answers are excellent responses for your problem. It is interesting that you had no contamination in your T-25 or agar plates.
I have found that without a cell layer to guide you, it is quite difficult to locate the media in a flask. Are you absolutely sure that you were looking at the media and not the bottom of the flask - the patterns in your picture resemble scratches and divots on the outside of the flask.
In any case, to help prevent contamination in your media, you can aliquot into 50 ml tubes and use those instead of an entire bottle at the same time.
Hope this helps!
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I have to treat cell line for 6 days with compound to see its deleterious effects. But the thing is within 48 hours cells got confluenced. So how to do this experiment? Should I just decrease serum concentration or any other way to complete this type of study?
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We have done a few experiments where we treat for six months. We seed at a lower density and let the cells grow (in treatment) for 5 days (they are near confluent) then trypsinize cells taking a portion of the cells to continue the treatment with and re-treating. However we have found some strange things happening over the course of 6 months. Looking back we realize that we have selecting for cells with a growth advantage. Problem is 1)this does not likely to reflect actual exposure in vivo and 2) we have possible/probably diluted the affected cells that are of more interest.
Does anyone have any thoughts?
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I have been working with 3d spheroids for a few months. I started with HCT-116 colon cancer cell line and have not had any problems, this cell forms tightly aggregated spheroids. However, I have not been able to form spheroids from HT-29 cell line. I've tried different agarose concentrations (1-3%) and different cell concentrations (800-10.000 per well) and this cell line won't form the spheroids, just cell clumps. Does anyone have any idea what could be happening? I've seen in many papers spheroids from HT-29 and theorically they are formed easily. I use the following protocol for the HCT-116:
96 well plate flat bottom coated with 50uL of 1.5% agarose. I plate 2000 cells and centrifuge 1000rpm for 5 minutes. Then, I incubate for 4 days untill the spheroids are formed.
Thanks in advance.
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Dear Gabriel,