Cell Line - Science topic

Hi,

Here are a few solutions I hope they will work:

1. Reduce the trypsin exposure time. Instead of 2-3 minutes, try monitoring under a microscope and stop as soon as the cells detach (usually under 1-2 minutes).

2. Avoid directly placing thawed cells in T75 flasks. Instead, seed them at a higher density in a smaller vessel (e.g., T25 or T12.5) for the first passage to encourage better recovery.

3. Perform a mycoplasma test to ensure your culture is clean.

4. Try obtaining a fresh vial of PC3 cells from the ATCC or another reliable source.

5. Try changing the media more frequently (e.g., daily instead of every 2 days).

6. Ensure media preparation and storage conditions.

i use a different method to grow spheroids. What I have done is grown cells (5000/well) in 96-well plates coated with 1.2% agarose. Because of the plate size, I usually would need to change growth media or treatments every 48 hours. I’ve realised that the handing of the spheroids (Method) is what contributes to variations in shapes etc. If you use a lot of force when adding fresh media, tilt the plate, or pipette too close to the spheroids, that usually causes it to disintegrate, flip on on side and change shape.

1. Searching on website ATCC: atcc.org https://www.atcc.org/, for more gene/target details;

2. Or uniprot website: https://www.uniprot.org/, you may identify the conresponding protein's function;

3. ECM related stroma cells, incluidng myofibroblast, endothelial cells may helpful.

Regarding your research questions:

Should you study tumor cells (lung cancer)? Yes, studying tumor cells is highly relevant, especially since your gene of interest is potentially contributing to lung cancer. It’s important to understand the gene’s role in the context of the primary disease you’re investigating.

Should you study immune cells or connective tissues? Given that your gene is highly expressed in lymphoma and leukemia and is associated with an enzyme and ECM, it might be particularly relevant to study its role in immune cells, especially since the immune system plays a significant role in lung cancer progression. Additionally, since the gene is ECM-associated, studying its role in connective tissues such as CAFs could provide insights into how it might affect tumor growth, invasion, and metastasis.

No specific study was identified that used BV2 cells treated with fish oils dissolved in hexane for an MTT assay. While research on MTT assays with BV2 cells and the general cytotoxicity of fish oils exists, the method of dissolving fish oils in hexane as a stock solution was not explicitly documented in the resources reviewed.

However, studies have addressed the utility and challenges of the MTT assay in measuring cytotoxicity and metabolic activity, emphasizing the importance of proper solvent use to avoid cytotoxic effects unrelated to the treatment itself. Organic solvents like hexane should be carefully evaluated to ensure they do not interfere with assay outcomes or cell viability independently​

MDPI

​If you're considering using hexane, it would be prudent to run a preliminary control experiment with cells treated with hexane alone to determine its impact. Let me know if you'd like assistance in designing such an experiment!

These cells are transformed and immortal. They probably went through 100s of passages before the company got a hold of them. It does not matter.

Hello Akshaya Nagarajan,

The two most common methods for protein quantification are based on either mass spectrometry or immunological techniques like Western Blotting.

Western Blotting is often used for protein identification or relative quantification. It can also be used for absolute quantification if appropriate calibration standards are used.

Mass spectrometry based techniques offer superior data quality and reproducibility. It can achieve low limits of detection, provided that technical pitfalls, such as incomplete protein extraction, incomplete proteolysis, and artifactual protein modifications, are appropriately controlled and considered.

You may use selected reaction monitoring (SRM), also referred to as multiple reaction monitoring.

A Western blotting assay essentially depends on the specificity of the antibody used. In contrast, an SRM assay depends on multiple parameters, such as the retention time, the mass-to-charge ratio of the precursor ion and selected fragment ions of the targeted peptide, and the relative signal intensities of the detected fragment (transition) signals.

These values are then weighted and combined to derive a score that indicates the probability that the targeted peptide has been detected.

The paper attached below will be helpful.

Best.

CD8 is not expressed by macrophages. The only way to express this protein would be through gene transfer and with a suitable promoter (CD11b promoter for example).

Hi all,

Does anybody have this cell line (ENU1564)?

If yes, I hope you can share with us.

Thank you,

Mohammad

Here are a few strategies to improve stability:

1.Use a Stable Cell Line

2.Optimize Transfection Conditions

3.Monitor Promoter Activity

4.Check Media and Culture Conditions

Practical Tips:

▪︎Test different transfection methods if stability is inconsistent.

▪︎Perform clonal selection to isolate a line with stable luciferase expression.

▪︎Maintain a constant, controlled environment to reduce variability.

Hello,

my email: shirin_sh777@yahoo.com ,

Thanks a lot for your attention,

Best regards

5% pen-strep is too much for most of cell lines. It might cause toxicity due to cell stress and affect the growth. You may want to consider change it to 1%

Abhinav Kethiri, Gaussian and Normal distribution is the same. The model became famous after a description by Friedrich Gauss who derived it in the context of a system of normal-equations (a step to solve a system of linear equations). Therefor it's sometimes called the Gaussian and sometimes called the Normal distribution.

I know that GraphPad Prism offers "test for normality". However, these are almost useless for the purpose you used them. A hypothesis test checks if the observed data (the sample) already provides sufficient information about a particular aspect of a model. In the normal distribution test, the model is some continuous univariate distribution having a mean and a variance, and the aspect being tested is that the functional form of the distribution is Gaussian. This is a restriction of the more general model of "some continuous univariate distribution". The whole aim of the test is to reject this restriction, in which case one would have demonstrated that the observed data already provides sufficient information to "see" (clearly enough) that the hypothesized special case is not accounting for all aspects of the real world. If you are able for some sample to reject this hypothesis, you still don't know if the kind of deviation if of any relevance (irrelevant deviations will lead to rejection in large samples), and if you fail to reject it, also you don't have any use information, as the test may have overlooked an actually relevant deviation (relevant deviations will not lead to rejections in small samples).

Dissolving erythromycin: Dissolve the erythromycin compound in an appropriate solvent. Common solvents include DMSO (for water-insoluble compounds) or sterile water. Ensure that the concentration in the solution is appropriate for the subsequent MTT assay. Generally, using a smaller volume of solvent for dissolution allows for easier concentration control.

Filter sterilization: If the solution is to be used in a cell-based assay, it is recommended to filter sterilize it after dissolution, typically using a 0.22 µm filter, to ensure that the solution is sterile and suitable for addition to cell culture.

Dilution and concentration: Dilute the erythromycin solution using cell culture medium or PBS to achieve an appropriate concentration range. For the MTT assay, it is recommended to prepare different concentration gradients to evaluate their effects on cell viability.

MTT assay: Add different concentrations of erythromycin solution to MCF-7 cell culture and incubate for a period of time (usually 24-48 hours). Then follow the standard MTT assay procedure, add MTT solution, incubate for a period of time to dissolve the formed formazan, and read the OD value on a microplate reader (usually at 570 nm wavelength).

Data analysis: Calculate cell viability based on OD value, and draw a concentration-viability curve to determine the cytotoxic effect of erythromycin.

Hello Maha Shawqi

Please note that antibiotics in culture should be used at optimal concentrations. It should not be used as a replacement of sterile practice. Antibiotics are toxic to cell lines. Most primary or normal human cells show reduced growth rates in the presence of antibiotics. The antibiotics could affect the metabolism of cultured cells, cell proliferation, differentiation or gene expression, though little is known.

Keeping the cells free from microorganism contamination can be accomplished with proper knowledge of good laboratory practice. Following all the guidelines towards a sterile technique makes these compounds unnecessary. Aseptic techniques, including a sterile work area, sterile reagents and media, good personal hygiene and sterile handling, act as a barrier between bacteria in the environment and sterile cell culture.

Nevertheless, if you still wish to add antibiotics for culturing normal cell lines, which I would not recommend, it becomes essential that you first perform a dose-response test to determine the level at which toxicity begins and accordingly adjust the concentration of antibiotic.

The recommended antibiotic concentration for mammalian cell culture is 100 µg/mL (1% v/v). However, for primary cell culture, the concentration of antibiotic is slightly higher as the chance of contamination is high for the first few weeks. Antibiotics provide extra layer of protection from factors that can cause contamination.

Best.

May consider using Permai fluorescence dye.

Dear V H Krishna Prasad,

Check the following Institute.

National Institute of Pharmaceutical Education and Research

NIPER Ahmedabad,

Opposite Air force Station, Palaj

Gandhinagar-382355, Gujarat, India.

 +91 79 66745555 , +91 79 66745501

Their cell line repository consists of mammalian cancer cell lines, murine primary cell lines and non-cancerous cell lines. I am not sure whether you will succeed but you can always try. lf you are interested, MM.1S cell line is available at ATCC

Best.

Hello Mahsa Daneshmand

The viability for most cells declines and reaches a nadir at 24 hours post-thaw. Most, if not all, of this decline appears to be due to apoptosis (as opposed to necrosis) induced by the stress of the cryopreservation process. After this time point, cells begin to recover and enter exponential growth. Give the cells a day or two more to recover from stress. Usually, 24 hours should be sufficient for adherent cells to attach to the substratum after thawing.

Also, you mentioned that you have used pre-warmed media, which is right because moving from a sub-zero environment straight to room temperature will put cells under a lot of stress. Therefore, it is important to make sure that your cells are put into pre-warmed media right after rapid thawing.

If you have provided an appropriate culture surface, then the most common cause for failure of cells to attach to the substratum is environmental stress. Stress on cells in culture is mediated by biophysical conditions, or by components that are either present or formed in the media. I hope you are using the right media for the culture of CHO. Ham’s F-12 was originally developed for the growth of CHO cells.

Finally, mycoplasma contamination may also be the cause of the problem because mycoplasma contamination can affect many properties of the cells such as function, growth, metabolism, morphology, attachment, and many other properties.

Best.

Neža Rugelj Jiarui Wang

Hello,

I am facing the exact same problem, my cells are alive in the T75 and T150 but die randomly in 24 well plates. I am culturing HDFs. I tried a different batch of plates and different coatings as well. Have any of you figured out a solution to this problem?

Thank you!

Thank you so much!

Hi Jasmin,

I prepared a stock of 1 mg/ml and kept in freezer.

I use 1:10000 dilution to induce the expression of my protein of interest.

Hello Giulia de Martino

You may centrifuge at 1200 rpm or (300 x g) for 5-7 mins for a viable cell count.

Best.

AR42J cell line

Data from RNA-seq and qPCR should correlate. qPCR has a wider dynamic range and higher sensitivity (provided the assay is well optimized), what may lead to generally higher LFC values in some cases - but still positive (or negative) LFCs from RNA-seq should correspond to positive (or negative) LFCs from qPCR.

Given the error bars show the standard errors, their size depends on 3 things: (1) the measurement precision (technical variance), (2) the biological variance, and (3) sample size. The technical variance may further have some hierarchical structure (replicates on the same plate, replicates on different plates etc).

You may have the possibility to reduce the technical variance by using a carefully optimized and standardized assay. The biological variance is what it is. The sample size is fully under your control: larger sample sizes will give smaller standard errors (under otherwise identical conditions).

I may be wrong but the videos look like it could be a fungal (yeast ?) contamination : has it gone away after freezing or persisting?

HAC15 cells will adapt to most sera, but their response to Angiotensin II and potassium changes significantly. Be aware that steroid production in HAC15 cells depends on the applied serum and HAC15 cells cultured in FBS produce other steroids than in CCS. For high aldosterone secretion, I recommend 2.5% UltroSerG serum. HAC15 cells cultured in FBS will divide quicker than in CCS.

Dear Vasco Branco ,

i am also looking for this cell line. Did you find a reliable provider? I would be very thankful to hear from you!

Thank you and best regards!

Thank you very much for answering Mu-Shen Chang

As you already proved that transfecting either plasmid A or B works separately, but only failed with co-transfection, it is recommend to transfect one time each but in a time-dependent manner. For example, transfect plasmid A, wait for at least 1 h than apply DNA-lipo mixture B or vice-versa and test each condition for the best condition. I also agree with other says, check your plasmids DNA quality before any downstream application, when nicked DNA become more than supercoiled form, it substantially increase transfection difficulty.

Best.

Hello Kushal Junghare

TPCK irreversibly inhibits the serine protease α-chymotrypsin. TPCK has been shown to specifically alkylate the histidine-57 moiety in the active center of chymotrypsin and chymotrypsin-like serine proteases.

Thus, TPCK is designed to react specifically with the active center of chymotrypsin, completely inhibiting the proteolytic activity of chymotrypsin with little effect on the activity of trypsin.

The use of TPCK is necessary during IAV infection because it will help to impair the activity of contaminating chymotrypsin, which does not promote IAV infectivity but can cause damage to cell monolayers.

Therefore, it is usually recommended to use TPCK-treated trypsin for IAV cultures in cell lines.

Best.

I’m facing the same problem with my mES cell lines when trying to transition them to 2i media

Hammad Mufti is right. MTT is cytotoxic. Check out on why the cells are not able to reduce MTT within 4 hours. Maybe the culture conditions that alter the metabolism of cells could be affecting the rate of MTT reduction.

Dear Dr. Munmun Kumari

The human alveolar type II cells are known for their role in innate immunity. They express surface molecules necessary for efficient antigen presentation. A549 which is a type II alveolar epithelial cell line, express cell adhesion molecules (CAMs) on the surface when infected.

As you mentioned that A549 cells experienced bacterial contamination twice, could be the reason for the abnormal phenotypic behavior. Longer time taken for trypsinization could be because of the increased expression of CAMs on the cell surface that experienced bacterial infection twice. Please note that CAMs help cells stick to each other.

As Ciaran Daly mentioned, you need to purchase a fresh vial of A549 cells.

Just a suggestion. Whenever cells get contaminated in culture, they should be discarded at the earliest. Even if you succeed by treating the contaminated cells with antibiotics, they are not fit for further culture and cell-based assays because they will no longer show the original characteristics.

I have attached a few references below which you may want to refer.

Regards,

Malcolm Nobre

Malcolm Nobre Many thanks!

Hello Vandana,

1. These black dots do indicate contamination, and they are not a byproduct of cell metabolism. They are attached to the plate surface and that is why, washing reduces their number but can't get them rid of completely. Did you check them under high magnification to see if they are motile?.

I do recommend to discard these cells and thaw the back up vial, if you have any. If not, wash with PBS harshly once every 1 hr for one day and do a single cell dilution.

2. The cells definitely look stressed.

Good luck.

Hello Sanjay Kumar,

The cell lines aren't recommended to transport at -20 degree C as it will kill the cells due to the cold injury. The addition of cryoprotectants may not help as well. We have developed a new live cell shipping solution that can be used to ship cells at ambient temperature for 5-7 days. It is simple and the cells can be shipped inside a small bubble mailer via regular courier thereby reducing the shipping cost significantly. Please reach out to sjoseph@pharmaplanter.com or www.pharmaplanter.com for more details.

Hello Antra ..

It would be difficult to answer your question. Cell lines respond differently to puromycin selection. In mammalian cells, the optimal level of puromycin is typically around 1µg/ml. But I suggest that you determine the optimal puromycin concentration for your cell line before initiating your experiment.

You may follow the protocol provided below.

1. On day 1, plate the oral carcinoma cells in ten 60mm petri dish and grow at 37°C, 5% CO2 overnight.

2. On day 2, when the cells are approximately 80-85% confluent, add puromycin (diluted in culture media) at a final concentration of 1-10μg/mL in 1μg/mL increments to the cells and label the petri dish (1-10).

3. On day 3, examine the cells each day and change to fresh puromycin-containing media every other day.

4. The minimum concentration of puromycin that results in complete cell death after three to five days is the concentration that should be used for selection in your experiments. If you wish, you may repeat this titration with finer increments of puromycin to determine a more precise optimal puromycin concentration.

I am also attaching another protocol which will help.

Best.

I personally received Mtt assay

if you are interested in learning about the basic Of this assay you can read this article

(( Proinsulin C-Peptide Enhances Cell Survival and Protects against Simvastatin-Induced Myotoxicity in L6 Rat Myoblasts))

Best Wishes.

Hello Elysha Nur Ismail

Insulin stimulates the growth and proliferation of a variety of cells in culture. In cell culture, it interacts synergistically with other hormones and growth factors such as PDGF, FGF, EGF, tumor-promoting phorbol esters, and thrombin, to stimulate progression through the cell cycle. In some cells, the growth-promoting effects of insulin appear to be mediated primarily by its low affinity interaction with receptors for insulin-like growth factor I (IGF-I), while in others, insulin appears to stimulate growth by binding to high affinity insulin receptors.

The insulin and IGF-I receptor proteins, like the receptor proteins for other growth promoting hormones such as EGF and PDGF, are closely associated with tyrosine-specific protein kinase activities. The mechanism by which the binding of insulin to its receptor and activation of the receptor-associated tyrosine protein kinase activity control intracellular protein phosphorylation and dephosphorylation reactions, help in the passage of cells through the phases of the cell cycle.

So, insulin forms an important constituent of the growth media that can help in the optimal growth of FHs74 cells. If you do not add insulin in the media, you may not observe optimal growth. If you are planning to use these cells for cell-based assays, they must be in good condition otherwise you may end up with unreliable results.

Best.

Dear Dr. Igor Andrade Santos

How about trying HT-29 colon cancer cell line? It has been shown to offer high transfection efficiency, making them suitable for gene expression and regulation studies.

To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. Add complete growth medium and aspirate cells by gently pipetting.

Regards,

Malcolm Nobre

Hello Abraham Castro-Cruz!

It is possible that you diluted the cells too much during the passage. Try planting them at a higher concentration (density) next time.

Best wishes,

Rustem Uzbekov

Hello Subash,

If you are still working on the electroporation of your cells using Neon® Transfection System, here are some helpful references you could check:

Thank you for your responses ...

- Cells are probably stressed due to freezing procedures.

- Even cells look abnormal, but they continue to proliferate!

- I've tried to culture them on small surfaces, even for adherent cell it's hard to form a colony (confluence) and look abnormal under microscope.

Hi Cristina,

When you say invasive, it is not clear if you mean a cell line derived from an invasive melanoma, or one that shows invasion in an assay (in vitro or in an animal).

But anyway, on the whole, the more invasive the melanoma from which a cell line was derived, the less pigmented it is in culture. Most pigmented human melanoma lines are from primary tumours. However, there are a few exceptions, such as this one:

https://www.nature.com/articles/s41598-017-02197-y

Meenhard Herlyn's lab in the Wistar Institute (Philadelphia) also keep a large library of human melanoma lines, and they can surely help you with finding what you want.

Sumit Upadhyay The white substances appearing in your ARPE-19 cell cultures could be due to several factors, such as cell debris, protein or reagent precipitation, fungal or bacterial contamination, or issues with the culture medium. To address this, examine the substances under a microscope, check the quality of your culture medium and reagents, ensure that culture conditions are optimal, and consider subculturing the cells into fresh media. If the problem persists or is unclear, consulting with a colleague or cell culture expert may provide further guidance.

Hi Andreia Peixoto, I totally agree with . Could be useful to test in a transient transfection the overexpression of your POI just to check the functionality of your vector and promoter of choice.

Then what came to my mind since you said it happened twice with 2 POI in the same vector could be a vector-related issues.... Could it be that only the Puromycin expression cassette got integrated? Are there some highly homologous sequence surrounding it? It would be useful to have more info about the vector you are using...

Let us know, I'm getting curious! Good luck!

Try the WB for detecting the LC3B-II or the IF for detecting the LC3B-II puncta.

Please contact us through our company website.

Hello Chuancong Zhu

p53 of ES2 cell line is mutant.

Please refer to the links attached below for more information.

Best.

Dear Colleague,

I hope this message finds you well. Determining the optimal puromycin concentration for selecting non-small cell lung cancer (NSCLC) cell lines transformed with lentivirus requires an initial kill curve experiment to identify the lowest concentration that effectively kills untransfected cells while sparing the transfected ones. Here is a detailed and logical approach to determining the best puromycin concentration:

Steps to Determine the Optimal Puromycin Concentration

Example Protocol

By following these steps, you can accurately determine the best puromycin concentration for selecting NSCLC cell lines transformed with lentivirus, ensuring effective selection of transduced cells while minimizing toxicity.

Should you have any further questions or require additional assistance, please feel free to reach out.

This protocol list might provide further insights to address this issue.

I think it is only right to treat each control as respective control for each plate.

Treating monolayer culture cell lines with essential oils involves several critical steps due to the hydrophobic nature of these oils, which affects their solubility and distribution in aqueous culture media. The process begins with preparing the essential oil solution using solvents like DMSO, emulsifiers such as Tween 20 or Tween 80, or cyclodextrins to increase solubility and ensure even dispersion within the culture medium. The concentration of the essential oil must be carefully optimized through dose-response studies to avoid cytotoxicity while achieving the desired biological effect. During incubation, standard conditions should be maintained, and gentle agitation may be necessary to ensure the oil remains uniformly distributed. Post-treatment, cellular responses should be evaluated using assays like MTT or XTT, with appropriate vehicle and positive controls included to validate results. Detailed documentation of experimental conditions is crucial for reproducibility, and repeating the experiment in different conditions can confirm the consistency of the findings. This approach ensures that the treatment is effective and the data obtained are reliable.

For AGS cells, the optimal concentration of DharmaFECT Duo to minimize cytotoxicity while ensuring effective transfection typically falls within a certain range, depending on the specific conditions and cell line characteristics. Generally, a good starting point is to use a concentration between 0.05% to 0.2% of DharmaFECT Duo in your transfection mixture.

However, to determine the exact optimal concentration for your specific experiment, it's crucial to perform a preliminary cytotoxicity test. This involves transfecting the cells with varying concentrations of DharmaFECT Duo and assessing cell viability 24 to 48 hours post-transfection using a cytotoxicity assay such as MTT, XTT, or a similar viability assay.

Here is a basic protocol outline for the preliminary cytotoxicity test:

1. Prepare Serial Dilutions: Prepare a series of DharmaFECT Duo dilutions (e.g., 0.05%, 0.1%, 0.2%, 0.3%, and 0.4%).

2. Transfection: Transfect AGS cells with these different concentrations while keeping other variables constant (e.g., amount of siRNA or plasmid DNA).

3. Incubation: Incubate the cells for 24-48 hours.

4. Assessment: Use a cytotoxicity assay to evaluate cell viability at each concentration.

5. Determine Optimal Concentration: Identify the concentration that provides effective transfection with minimal cytotoxicity.

Adjust as needed based on your specific experimental conditions and requirements.

3T3-L1 and H9C2 cell lines are not typically used for atherosclerosis models. Here is a brief overview of these cell lines and their typical applications:

Atherosclerosis Models and Suitable Cell Lines

Atherosclerosis research often involves the use of endothelial cells, smooth muscle cells, and macrophages, as these are the primary cell types involved in the disease process. Commonly used cell lines for atherosclerosis studies include:

Inducing Atherosclerosis-like Conditions

If you are limited to using 3T3-L1 cells, here are some suggestions on how to adapt these cells for atherosclerosis-like studies, particularly focusing on aspects related to lipid metabolism and inflammation:

Assessments and Measurements

To evaluate the induced atherosclerosis-like conditions in 3T3-L1 cells, you can perform the following assays:

Conclusion

While 3T3-L1 and H9C2 are not ideal for direct atherosclerosis studies, you can adapt 3T3-L1 cells to study certain aspects of lipid metabolism and inflammation that are relevant to atherosclerosis.

I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?

A highly recommended rabbit-produced antibody for immunoprecipitation (IP) and subsequent western blotting of FLAG-tagged proteins in mouse-derived chondrocyte cell lines is the Anti-FLAG® M2 antibody from Sigma-Aldrich. This antibody demonstrates high affinity and specificity for FLAG-tagged proteins, making it suitable for capturing and detecting these proteins in various applications, including IP and western blotting [1][2][3][4]. The Anti-FLAG® M2 monoclonal antibody is widely used due to its ability to bind both N-terminal and C-terminal FLAG tags without requiring calcium-dependent conditions, ensuring reliable and consistent results [5][6].

[1] Brizzard, B., Chubet, R., & Vizard, D. (1994). Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution.. BioTechniques, 16 4, 730-5 .

[2] Knappik, A., & Plückthun, A. (1994). An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments.. BioTechniques, 17 4, 754-61 .

[3] Ferrando, R., Newton, K., Chu, F., Webster, J., & French, D. (2015). Immunohistochemical Detection of FLAG-Tagged Endogenous Proteins in Knock-In Mice. Journal of Histochemistry & Cytochemistry, 63, 244 - 255.

[4] Verhagen, A. (2006). Using FLAG Epitope-Tagged Proteins for Coimmunoprecipitation of Interacting Proteins.. CSH protocols, 2006 5.

[5] Itakura, E., Chen, C., & Bono, M. d. (2017). Purification of FLAG-tagged Secreted Proteins from Mammalian Cells.. Bio-protocol, 7 15.

[6] Zhang, L., Uder, S., Juehne, T., Brizzard, B., & Song, K. (2002). Nonradioactive assay of FLAG-tagged MAPK using ANTI-FLAG antibody-coated multiwell plates.. BioTechniques, 32 2, 442-7 .

The typical procedure involves dissolving oil-based compounds in solubilizers like glycerin, Tween 20, or DMSO, which allows them to easily mix with DMEM. It is standard practice to evaluate the cytotoxicity of the solubilizer alone to ensure it does not affect cell viability.

It would be helpful if you could specify the drug you are researching.

The issue might be quite simple. It's possible the stock drug has degraded and lost its effectiveness what affected all dilutions. Additionally, there could have been errors in your calculations or when preparing the dilutions. Have you attempted to repeat the experiment using freshly prepared dilutions?

Thank you Paul Rutland and Jeeva Sasikumar . I've just received the negative control vial from the manufacturer and it was the first time I opened it, so I don't think a contamination is possible. But I'll keep that in mind!

Hello Hyun-Keun Kim Francesc Estrany ,

sorry if i was not clear.

I have mRNA expression in both cell lines, but protein expression in only one cel line.

Hi, did you find any solution for that problem ?

Hello, if you still need some input feeel free to contact us. Best, Fabian

I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?

To explore the Angiotensin-Converting Enzyme (ACE) and Carbonic Anhydrase 1 (CA1) pathways for blood pressure regulation, consider using the following cell lines in addition to CaCo2, HUVEC, RAECs, and HEK293:

These cell lines provide a comprehensive platform for investigating the mechanisms underlying blood pressure regulation through the ACE and CA1 pathways.

[1]Chen, L. Q., Howison, C., Spier, C., Stopeck, A., Malm, S. W., Pagel, M., & Baker, A. (2015). Assessment of carbonic anhydrase IX expression and extracellular pH in B-cell lymphoma cell line models. Leukemia & Lymphoma, 56, 1432 - 1439.

[2]Chen, R., Suchard, M., Krumholz, H., Schuemie, M., Shea, S., Duke, J., Pratt, N., Reich, C., Madigan, D., You, S., Ryan, P., & Hripcsak, G. (2021). Comparative First-Line Effectiveness and Safety of ACE (Angiotensin-Converting Enzyme) Inhibitors and Angiotensin Receptor Blockers. Hypertension, 78, 591 - 603.

[3]Altamimi, S., Khalil, A., Khalaiwi, K. A., Milner, R., Pusic, M. V., & Othman, M. A. (2010). First-line drugs for hypertension. São Paulo Medical Journal, 128, 47 - 47.

I should have read about U6 promoter before having this question. U6 promoter is not likely to be used for long transcripts as I am considering. I think I may continue using empty lentiCRISPRv2 for Cas9 expression. Just don't know why my cells get stressed after transduction and nearly stop growing, perhaps because it is a primary cell line.

you should always do a minimum killing assay to find out the minimum concentration of puromycin to kill all the cells.

From my personal experience, epithelial cells are very sensitive to puromycin, and i used only 0.5ug/ml of puromycin to kill all my epithelial cell lines. For many other cell lines, i have seen 1-10ug/ml of puromycin being used.

I would suggest you set up a preliminary assay to test the 0/5. 1, 2, 4, 6, 10 ug/ml of puromycin to find out the minimum concentraion to use in your cell line.

Thank you very much for this comprehensive answer !

As long as you have data at >50% inhibition, you can calculate the IC50 using the Hill equation, allowing the maximal % inhibition to be varied as a parameter, instead of being fixed at 100%.

A possible reason for not being able to exceed 65% inhibition may be that the compound is not soluble enough.

I think what you are asking is can you take PBMC from two different individuals and incubate them together as a in vitro model for graft versus host disease? Yes. That is known as a MLR - mixed lymphocyte reaction. Typically, one set of PBMC is inactivated by irradiation or mitomycin C treatment first [this is called a one-way MLR]. You can use this to test the effect of adding compounds into the MLR to see if it affects the response.

Hello Haya,

You should seed cells in a minimum volume of 100ul per well in a 96 well plate.

Suppose you want to seed 1000 cells per 100ul per well, then for 100 wells (always consider extra wells), you will require

1000 x 100 = 100,000 cells in 10000ul (10ml).

Then from 10ml cell suspension, you pipette out 100ul per well which will give you 1000 cells/100ul/well. Always pipette the cell suspension up and down a number of times so that each well will receive equal number of cells.

If you need to seed just one well, then you make a cell suspension for 5 wells (4 wells extra) because pipetting small volumes can lead to pipetting error. So to avoid this error you should increase the volume. So for 5 wells, you will require 5000 cells in 500ul. Then pipette the cell suspension up and down and add 100ul to one well.

Best.

Hi Waqas, kindly check this- https://www.mdanderson.org/documents/core-facilities/Functional%20Genomics%20Core/Virus%20titering%20protocols.pdf