Melanoma - Science topic

Hello Melissa Thone

You should keep cancer cells on ice to maintain the viability of the cells temporarily. You should inject cells as quickly as possible after preparation because viability slowly decreases over time, even on ice.

You should use a 25g 5/8” needle for subcutaneous application.

For i.p. injections of adult mice, you may use 26 g 1/2” needle, for i.v. injections, you may use 26-27g 1/2” needle and a 30g 1/2” needle for some orthotopic injections.

You need to know that the higher the gauze number, the smaller the diameter of the needle. This will then exert more stress on the material and any cells in it. High shear stress and pressure can decrease cell viability.

The lower the gauze number, the stronger the needle, resulting in less chance of bending or breaking.

Hold a mouse with one hand, use the other hand to slide needle 5 to 10 mm subcutaneously and inject 50-100 µl cell suspension (Tumor cells - 1x10^5 - 1x10^6 cells). You should watch for appearance of a bleb. Failure to insert needle far enough will result in leakage of tumor suspension when mice massage the area after injection.

1. Do not let the syringe with cell suspension sit undisturbed on bench for more than 5 minutes because the cells can settle quickly.

2. Be sure to resuspend cells well in the syringe barrel before each injection.

3. Do not pass the cells through needle more than once since this will shear cells and reduce total cell number.

Also, please refer to the article attached below for more information.

(See basic protocol 1)

Best.

The media looks normal with pinkish color Not cloudy at all. I use 10% DMEM and add 1% pen/strep for the culture media.

Scientists have developed a new method that can easily create immortal human mammary epithelial cells. The cells could greatly facilitate the examination of cell immortalization as it actually occurs during cancer progression. Every day, some of your cells stop dividing, and that's a good thing.

I am sharing a pdf, it would be very helpful for you.

PD-1 expression on naïve T cells is induced upon TCR activation. This transient expression decreases in absence of TCR signaling but is maintained upon chronic activation with a persisting epitope target. You used Human TransACT "Miltenyi Biotec", it may create a chronic activation on CD8+ Tcells. May be this activation results in lower expression of PD-1. Good luck

Has Mrs. O'Brien ever read my answer? Please let me know

If you know the source of the autofluorescence and it does not overlap with your targeted flourophore then you could use sequential scanning and tune the detector of the confocal to isolate your target and the autofluorescence in order to separate them. Confocal microscopy is always the better bet in cases of autofluorescence over standard bandpass filters of a fluorescent scope.

Hello Petra Fodor

I have not worked with MM28 cell line. But generally, while freezing cells the main points to remember in order to get high cell viability on thawing are:

1. Freeze cells when they are 80% confluent.

2. Carry out gradual freezing of cells i.e., -1 degree C/minute before you place your cells in liquid nitrogen. You may use Mr. Frosty freezing container commercially available that will help in gradual cooling. Place the freezing vials in Mr. Frosty container in -70°C freezer for a minimum of 4 hours and then remove frozen tubes and place them in liquid nitrogen (vapor phase) for long-term storage. Always use pre-cooled freezing vials. Freeze approx. 2 million cells per ml per vial.

3. MM28 cells are frozen in 90% FBS and 10% DMSO as the freezing medium.

Best.

Hello Md Marufur Rahman

RPMI 1640, based on the 5A Medium (developed by McCoy et al. 1959) and modified for the long-term culture of peripheral blood lymphocytes, is characterized by low levels of calcium and magnesium and high levels of phosphate. Multiple media were developed on the path to this medium (e.g., RPMI 1629, 1630, and 1634). It is widely used as a medium for suspension cultures; for example, of white blood cells, lymphocytes, and hybridomas. Please refer to the article below for more details.

I would recommend the use of DMEM+10% FBS for A375 melanoma cell line as mentioned in the ATCC website.

Best.

As to the first part of your question: the answer is no: there are currently about 1200 non-human primates cell lines in the Cellosaurus and none of them is from a melanoma. This is not surprising as this is a very rare disease in monkey. See for example:

Where it is stated that searches in a number of bibliographical databases "did not reveal any published report of uveal melanoma in a nonhuman primate."

and

Where it is said that "To our knowledge, this is the first documented case of melanoma in a cynomolgus monkey."

So its unlikley someone has had the opportunity to establish a non-human primate melanoma cell lines.

Best

Amos

PS: Disregard the 1st answer: there are a number of companies distributing cell lines that have people answering any question on cell lines by pointing to their catalog even if it is not relevant. AcceGen is not distributing any monkey melanoma cell line

By decreasing extracellular acidity (e.g. bicarbonate) and decreasing intracellular alkalinity (e.g. amiloride) you will substantially decrease migration.

Another useful drug is dasatinib that inhibits Src phosphorylation and limits the cytoskeleton activity involved in migration.

Dear Malcom,

Thank you very much for your informative answer. I did same as you mentioned with normal HDF along with a panel of cancer cells.

Thank you again.

Regards

Menuja

I hope this email finds you well.

I would like to invite you to have a look on the following links.

SSO: https://www.mathworks.com/matlabcentral/fileexchange/92150-sperm-swarm-optimization-sso

HSSOGSA: https://www.mathworks.com/matlabcentral/fileexchange/92130-hssogsa?s_tid=srchtitle

MOSFP: https://github.com/sh7adeh1990/MOSFP

Thank you very much for your time and efforts.

Dear Александр Отченашенко

The following PLCO Melanoma dataset(s) are available for delivery on CDAS. For each dataset, a Data Dictionary that describes the data is publicly available.

for more info: https://cdas.cancer.gov/datasets/plco/11/

Kind Regards

Hello Hanna Moalem

Besides lactate levels, you can try measuring other proteins. Please refer to the research article below for more information.

Nutrient deprivation has been shown to cause cancer cell death. To exploit nutrient deprivation as anti-cancer therapy, we investigated the effects of the anti-metabolite 2-deoxy-D-glucose on breast cancer cells in vitro. This compound has been shown to inhibit glucose metabolism. Treatment of human breast cancer cell lines with 2-deoxy-D-glucose results in cessation of cell growth in a dose dependent manner. Cell viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide conversion assay and clonogenic survival are decreased with 2-deoxy-D-glucose treatment indicating that 2-deoxy-D-glucose causes breast cancer cell death. The cell death induced by 2-deoxy-D-glucose was found to be due to apoptosis as demonstrated by induction of caspase 3 activity and cleavage of poly (ADP-ribose) polymerase. Breast cancer cells treated with 2-deoxy-D-glucose express higher levels of Glut1 transporter protein as measured by Western blot analysis and have increased glucose uptake compared to non-treated breast cancer cells. From these results we conclude that 2-deoxy-D-glucose treatment causes death in human breast cancer cell lines by the activation of the apoptotic pathway. Our data suggest that breast cancer cells treated with 2-deoxy-D-glucose accelerate their own demise by initially expressing high levels of glucose transporter protein, which allows increased uptake of 2-deoxy-D-glucose, and subsequent induction of cell death. These data support the targeting of glucose metabolism as a site for chemotherapeutic intervention by agents such as 2-deoxy-D-glucose.

I hope this helps.

Best.

Interested

Hello Mohammad Nasfikur Rahman Khan

When cancer is initiated and starts growing, the tumor tissue requires nutrients and oxygen because they are rapidly dividing. It is this requirement of the tumor that causes stimulation of angiogenesis. The angiogenic stimulators like VEGF are released by the tumor cells which in its presence endothelial cells will proliferate and migrate, eventually forming tube structures resembling capillaries. VEGF causes a signaling cascade in endothelial cells. Thus, formation of new blood vessels will satisfy the nutritional and oxygen requirements of the growing tumor. The growing tumor can invade the surrounding tissue with the help of matrix metalloproteinases that participate in the degradation of ECM components, including the basement membrane and the tumor surface, resulting in tumor cell migration into the nearby tissue. Thereafter the cancer cells undergo intravasation into the surrounding vasculature or lymphatic system and survive in circulation, and then undergo extravasation from vasculature to secondary tissue to form secondary tumor, a process known as metastasis.

Thus, the relationship between angiogenesis and metastasis is that the tumor micro vessel density is directly related to increased metastatic potential and therefore leading to poor survival in all forms of cancer.

I hope this is helpful.

Best.

My mother (who lived over 100 years) religiously stayed in the sun every day for 20 minutes, but not when the sun is very hot (in the afternoon), preferably in the morning. She used to say that a bit of the sun made her feel better as compared to cloudy days with no sun. She decided the 20 minute interval from experience.

X axis are gene expression values. Left y axis are correlation coefficient, right y axis is metastasis score

Your problem is with the ffpe method. Make sure the steps are repeated with precision materials. However, you can use other methods such as trisol to extract RNA and phenol / chloroform to extract DNA. The ctab method is used to extract the plant genome due to the high amount of carbohydrates and to extract the genomes of some bacteria for the molecular work of typing. In my opinion, repeat the paraffin removal steps again and use more Poly A due to the low amount of tissue and genome in the paraffin tissue used.

It depends on the promoter strength of neoR gene and the copy number of the overexpression construct with a cell. The gold standard is 0.4mg/ml.

For frozen section usually 4% PFA is a good fixative for overnight fixing followed by serial sucrose treatments. Sucrose treatment suppose to cryopreserve the cells/ prevent from damaging your cells from negative 80. If you do formalin fixing, then you usually have to do antigen retrieval (heat retrieval for example) in order to expose the antigens. Also, have you dried out your tissue during the IHC procedure? If you dry out your sections during IHC, tissue could look like this! try avoiding drying out your sections except the initial tissue adhesion into slide glass before washing off OCT from the tissue.

4T1 cells (mouse)

The International Skin Imaging Collaboration: Melanoma Project is an academia and industry partnership designed to facilitate the application of digital skin imaging to help reduce melanoma mortality. When recognized and treated in its earliest stages, melanoma is readily curable. Digital images of skin lesions can be used to educate professionals and the public in melanoma recognition as well as directly aid in the diagnosis of melanoma through teledermatology, clinical decision support, and automated diagnosis. Currently, a lack of standards for dermatologic imaging undermines the quality and usefulness of skin lesion imaging. ISIC is developing proposed standards to address the technologies, techniques, and terminology used in skin imaging with special attention to the issues of privacy and interoperability (i.e., the ability to share images across technology and clinical platforms). In addition, ISIC has developed and is expanding an open source public access archive of skin images to test and validate the proposed standards. This archive serves as a public resource of images for teaching and for the development and testing of automated diagnostic systems.

Did you try to grow this contaminant on mycological media, such as Sabouraud dextrose agar, or Pal sunflower seed medium? If you find any colony suspected as fungus, try to prepare wet mount preparation in Narayan stain to study the morphology. We have developed Pal sunflower seed medium in 1980 and Narayan stain in 1998 for the study of fungi. Once you are sure that contaminant is a fungus, you should try to fumigate your tissue culture laboratory and also add broad spectrum antifungal antibiotics to the cell culture medium. Such problem may occur when we work with tissue culture.

Hi Jonathan,

Here you have some publicly available datasets (free registration/acces request needed):

- US National Cancer Institute: https://cdas.cancer.gov/datasets/plco/11/

- US Center fo Disease Control and Prevention https://www.cdc.gov/cancer/uscs/public-use/obtain-data.htm

- Melanoma UK Dataset https://vitaccess.com/melanoma-uk-dataset

I hope this helps,

Regards

Dear Mariana Nunes,

Photoacoustics (PA) means the study of vibrations induced in matter by light. Laser light causes localized heating when it is absorbed by a surface. This in turn makes the target area expand and sends a pressure wave through the rest of the material. This effect enables imaging of opaque systems, particularly biological samples. Photoacoustics flow‐cytometry technique (PAFC) is based on non-radiative transformation of the absorbed laser energy into acoustic waves caused by the fast thermal expansion of the heated sample. This phenomenon is monitored through the changes in optical characteristics that are detected by an ultrasound transducer attached to the sample. For in vivo applications, PAFC either uses label‐free detection of cells with intrinsically light‐absorbing chromophores (e.g., hemoglobin, melanin, or cytochromes) or cell labeling with strongly absorbing dyes or nanoparticles as PA molecular probes. In general, PAFC is considered acoustic analysis, meaning the data collected and analyzed would be on sound waves. Although, PAFC is conducted using the same process as conventional flow cytometry, the sensor used is a transducer rather than light capturing sensors. Where photosensors convert light energy to electrical energy, transducers convert sound waves to electrical energy.

Hope this helps.

Best - Pradyumna

Hi Heba,

I agree with Brian the human colon cancer cells (SW480) are obtained from ATCC (American Type Culture Collection, United States) or from Europe. So you should contact them via mail to provide you the instructions.

Here's the link of the company:

Hope this may help you

check this link:

I think Silvia Torchio means this could be the reason of the culture problem.

For selection 1 microgram/ml should work with your cell line in 3-4 days. After selection, change concentration to 0.5 microgram/ml. Note that cell killing works best when cell density is low (~50%).

Thanks Khadija Elboukhari for your excellent insight.

we have had success with the enzyme called "Liberase TM"

Use of viagra does not cause the development of melanoma.

Hii,

I have a Question regarding this.

Can you suggest me the protocol of tumor injection i.e any Research paper reference.?

Dear Ahmed do follow this link to a project i was involved in last year. There is a dermoscopy tool kit with links to lots of website, where you will find an abidance of images for every skin type and skin lesion:

https://www.rcgp.org.uk/clinical-and-research/resources/toolkits/dermatology-toolkit.aspx I hope this is helpful. Julie

Ana Santos You say these are cell lines so, perhaps there is a publication (even if very old) that characterized these lines and by reading their discussion you can get some additional info about how these lines form spheroids. In general, cells form spheroids in conditions of low adhesion to the plate, so you can try different plates (flat vs round bottom low adhesion) and observe the cell clusters. The best way I know of identifying spheroids is to watch the cells make them. So I would suggest to watch the cells closely for a few days.

Hope this helps

Victoria

Hi Ana,

I also tried to make spheroids from these cell lines, however for A375 and G361 the spheroids were not as tight and partially disintegrated when handling. I used liquid overlay method and cultered the cells in ultra-low attachment plates (96 well format with round bottom).

Here are some protocols that worked out well for me:

https://www.jove.com/video/55544/an-efficient-flexible-cell-aggregation-method-for-3d-spheroid

For SK-Mel-1 cells I didn't try, but to my knowledge they are a suspesion cell line, so growing as spheroids might not be feasable for this cell line.

Hope this helps :)

All the best, Verena

Michael Lee No, we have not tried Prolong Glass yet.

Hi

You can use the following protocol to calculate IC 50 for your drug. PDF attached for your reference.

Naive question here, but how do you know if you are subcutaneous or intradermal. We've recently injected mice with the intention of injecting s.c. However, 1/3 of the tumors are growing much more slowing. I have been told this may mean that the tumors were injected intradermally. Is there any way to prevent this? Thank you.

B16 variants require media with specific AAs to form melanin. Thus, in RPMI-1640 they loose the brown color. It is best to culture in the recommended media of MEM with additional NEAA and EAA. This maintains the melanin.

As mentioned B16 variants are adherent cells, but when they become acidic or nutrient deprived they form cell mats and also release cells from the plastic surface. Further, they form exosomes and appear to have a large number of floating cell fragments. Thus, a 2 to 3 time per week split is optimal. Also assure that the split results in about 20% confluence.

Hope this helps.

Hi Kátia Morais ,

I'm coming a bit late but I also have trouble with transferrin, I can't detect any signal after using the concentration given by the literature. Did you try to use it at a higher concentration on the HDF cells? Do you remember if it worked ?

I do thank you

Using ELISA techniques you can measure protein amounts but not protein activity.

This should be done via adequate functional and/or activity assays for your target protein.

Michele:

Pembrolizumab (Keytruda) in Melanoma:

Ascierto PA DR, et al. KEYNOTE-002 Part 3: Phase 2 randomized study of 1L dabrafenib (D) and trametinib (T) plus pembrolizumab (Pembro) or placebo (PBO) for BRAF-mutant advanced melanoma. ESMO 2018 Congress.

Carlino MS, Long GV, Schadendorf D, et al. Outcomes by line of therapy and programmed death ligand 1 expression in patients with advanced melanoma treated with pembrolizumab or ipilimumab in KEYNOTE-006: A randomised clinical trial. Eur J Cancer. 2018 09; 101:236-243.

Daud AI, Wolchok JD, Robert C, et al. Programmed Death-Ligand 1 Expression and Response to the Anti-Programmed Death 1 Antibody Pembrolizumab in Melanoma. J Clin Oncol. 2016 12; 34(34):4102-4109.

Eggermont AMM, Blank CU, Mandala M, Long GV, Atkinson V, Dalle S, et al. Adjuvant Pembrolizumab versus Placebo in Resected Stage III Melanoma. The New England journal of medicine 2018;378:1789-801.

Hamid O, Robert C, Daud A, Hodi FS, Hwu W-J, Kefford R, et al. 5-year survival outcomes in patients (pts) with advanced melanoma treated with pembrolizumab (pembro) in KEYNOTE 001. Journal of Clinical Oncology 2018;36:9516-

Hodi FS, Hwu WJ, Kefford R, et al. Evaluation of Immune-Related Response Criteria and RECIST v1.1 in Patients With Advanced Melanoma Treated With Pembrolizumab. J Clin Oncol. 2016 05 01; 34(13):1510-7.

Kluger HM, Chiang V, Mahajan A, Zito CR, Sznol M, Tran T, et al. Long-Term Survival of Patients With Melanoma With Active Brain Metastases Treated With Pembrolizumab on a Phase II Trial. Journal of clinical oncology 2018:JCO1800204.

Long GV, Schachter J, Ribas A, Arance AM, Grob J-J, Mortier L, et al. 4-year survival and outcomes after cessation of pembrolizumab (pembro) after 2-years in patients (pts) with ipilimumab (ipi)-naive advanced melanoma in KEYNOTE-006. Journal of Clinical Oncology 2018;36:9503-

Robert C, Ribas A, Hamid O, et al. Three-year overall survival for patients with advanced melanoma treated with pembrolizumab in KEYNOTE-001. Journal of Clinical Oncology 2016 34:15_suppl, 9503-9503.

Robert C, Ribas A, Hamid O, Daud A, Wolchok JD, Joshua AM, et al. Durable Complete Response After Discontinuation of Pembrolizumab in Patients With Metastatic Melanoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2018;36:1668-74.

Schachter J, Ribas A, Long GV, Arance A, Grob JJ, Mortier L, et al. Pembrolizumab versus ipilimumab for advanced melanoma: final overall survival results of a multicentre, randomised, open-label phase 3 study (KEYNOTE-006). Lancet 2017;390:1853-62.

What is the normalization based on?

I think differences in gene expression between tissues are tricky on two levels.

In the first place, there is the normalization issue. In my experience, if you test for differential gene expression between tissues, a large proportion of the transcriptome comes out DEG, and it all depends on the normalizations used. So for a given gene: do you consider its expression relative to the rest of the transcriptome? Dangerous, because highly expressed genes in the background may "dilute" your transcript of interest, even if stoichiometrically, it is "upregulated" relative to the genes it interacts with. Or do you normalize relative to housekeeping genes? It is known that housekeeping genes exhibit tissue-specific expression. Do you consider it relative to the genes it interacts with? Interesting, but requires a lot of prior knowledge...

Secondly, there is also the question of what it actually means to be differentially expressed between tissues. Transcripts (and/or their corresponding products) hardly ever function on their own. They interact with the rest of the transcriptome/proteome, causing feedback to gene expression etc. These "backgrounds" are drastically different between cell types. Also, the same amount of mRNA may lead to different amounts of protein depending on the status of the cell (presence/activation of ribosomes, tRNA,...). As a result, the impact of a change in the number of copies of a transcript may be enormous in one tissue, versus negligible in the other.

These are actually considerations that to a large extent hold true even when comparing gene expression between conditions in the same tissue, but obviously the more different your samples are to start with, the more exaggerated the effects will be. So, my two cents is: be careful, it's a dangerous comparison. I would rather try healthy lung vs lung cancer and healthy skin vs melanoma.

Also , you can measure cell sizes by use image j program.

Pakize Neslihan Taşlı

Thank you for the valuable response.

I am also using enzymatic activity measurement with mushroom tyrosinase but i need to do western blot analysis on melanogenic pathways. I obtained B16F10 cell lines, now I am moving with them.

Regards

In addition to all replies above, new stock of FBS might be the culprit. Some FBS stocks don't seem to work for some cell lines. 2.

Creo que esas serian las mejores

Hi Mariyan

This might help.

Because you mentioned that you did not purify the AAV virus, but simply freeze-thaw and filter it, then you of course got a lot of cell fragments and media components in AAV crude extract. Purified AAV viruses produce only very low immune responses and other side effects, according to current reported studies and clinical trials. But if you used a cude extract, tt is bound to induce very strong immune responses in mice，even just use a AAV-GFP control, which maybe the reason for the observed anti-tumour activity. Maybe you inject a freeze-thaw cell lysate, you would got the same result. I strongly suggest you to purify AAV with ultracentrifuge followed by dialysis. Hope these can help you.

You could find more information about AAV production and application in this website: https://www.genemedi.net/i/aav-packaging

If you have at your reach a microscopy that allows you to detect fluorescence, you can show an image where the fluorescent signal of the cell nucleus overlaps the fluorescent signal of your nucleus protein (DAPI+AF488). In the supplementary file, you can include the control image.

Hi,

You will find the ISIC 2017 challenge dataset (with the corresponding annotated masks) from:

Exactly :) My pleasure, best regards, Przemek

A noticeable change in cell morphology that associated with a cell growth arrest often results due to sub-optimal cell culture conditions. These conditions include, a change in cell density, culture medium (growth factors), cell culture flask/plate (a change in extracellular matrix-induced signaling), and sub-optimal trypsinization of cells. Other conditions (the list is long) are also known to alter the cell morphology. Therefore, if you have notes from your cell culture conditions, it should be possible to identify potential factor(s) that might have contributed to your observations.

Hi Lukas Peiffer ,

I'd recommend doing a search on BenchSci for published antibodies (https://landing.benchsci.com/academic)

Please see attached for more detail.

I hope this helps!

Maurice

We use NOD/SCID mice in our commercial A375 xenografts (http://altogenlabs.com/xenograft-models/melanoma-xenograft/a375-xenograft-model/) and they tend to work well, with stable growth curves as well. I would make sure your starting injection is done properly (we use matrigel with 1 million cells) and then monitor for any kinds of symptoms that wouldn't correspond with normal tumor growth.

Hello,

I wondered this; these papers might be of interest:

Kluger, N. (2016). Cutaneous and systemic complications associated with tattooing. La Presse Médicale, 45(6), 567-576.

This is the ResearchGate link:

I have not seen the full text of this 2008 paper, but it sounds relevant:

Kluger, N., Phan, A., Debarbieux, S., Balme, B., & Thomas, L. (2008). Skin cancers arising in tattoos: coincidental or not?. Dermatology, 217(3), 219-221.

Kluger, N. (2009). Skin tumors arising in tattoos: coincidental or upcoming public health issue?. Expert Review of Dermatology, 4(4), 313-315.

Very best wishes,

Mary

HI Adi,

Hope you are doing well, you might contact MD Anderson Melanoma department for the cell line you seek. If you have requirement for any particular cell line let me know I would ask around.

Thanks

For staining naive B cells that are not in the B cell follicle, use anti-IgD; for germinal center B cells in the follicle, use anti-FAS(CD95), anti-GL7, anti-PNA, either two of those should work; for follicular dendritic cells in the light zone of germinal center, you can use PE-conjugated-hapten(NP) in vivo, or anti-CD21; for T cells, use anti-CD4.

We used TG3 transgenic mice which are spontaneous development cutaneous melanoma. We want to get melanoma cells from this mouse line for further study.

I hope to clarify some of what you have written; most of it is correct, but there are some fundamental misunderstandings.

Most skin cancers do not spread and are not benign. Squamous Cell Carcinoma and Basal Cell Carcinoma, specifically know as Keratinocyte Carcinoma or generally referred to as Non-melanoma skin cancer are malignant, localized, seldom fatal, and are diagnosed more than every other cancer combined. Melanoma is very serious, but melanoma is not close to the largest number of incidents identified.

I'm not sure of the current status of publication, but I am aware of a person who purchased most of the apps available where they report the ability to distinguish benign vs. concerning features on the skin. The results are not generally favorable.

As someone who works with dermatologists while seeing patients, I am impressed with their ability to visually distinguish concerning growths from others. That said, it would seem teaching a computer to provide the same level of interpretation should be straightforward, but I have yet to see one that can outperform new dermatologists.

I hope that you are interested in this type of research and you are the person to develop the methodology to make visual identification accurate and reliable. I believe it is possible.

Hi Karel, I believe you may find useful the USPFT recommendations: https://www.uspreventiveservicestaskforce.org/Page/Document/RecommendationStatementFinal/skin-cancer-screening2

"The USPSTF concludes that the current evidence is insufficient to assess the balance of benefits and harms of visual skin examination by a clinician to screen for skin cancer in adults." (in asymptomatic adults)

Check out these two articles, they have very specific information regarding the cell culture media. Hope you can find what you are looking for.

For representative Melanoma cell lines, you should used melanoma cell lines mutated on BRAF (such as A375), NRAF (such as SkMel2), NF1 (such as MEWO) or "triple negative" (such as CHL1). Read this recent publications (Luebker-Oncotarget 2017) for details of genomic alterations in a wide range of melanoma cell lines.

Hi Ulrich, I would like to introduce you to the work of my colleague, Dan Adams, who is isolating giant cells from patient blood samples.

These cells bear the hallmark of both macrophages and megakaryocytes and are probably the tumor associated macrophages (giant cells) that pathologists described in biopsies decades ago.

They could be the missing link in the interactions of the immune system with cancer cells.

Daniel Lee Adams

Have a look at this article:

Like Pawan said may be you ought to check the knockdown using western blot. A375 cell lines are known to be suitable transfection host and you achieve good transfection starting at about 10nM of the target gene you want to silence using HiPerfect transfection reagent . Higher concentrations of the reagent ofcourse is toxic but you can incubate the mix for about 30 minutes as well.

Did you check the health of the cells before plating them or test them for mycoplasma if you don't see any other things that need to be taken care of

How about your siRNA quality?

And check if by mistake you have added the Serum in your mix because it will interfere the process

Try a 'mock' transfection experiment where you do not add the siRNA or the scrambled RNA. Than you can exclude, that your cell-treatment causes the change in mRNA-amount or protein production.

Hope it helps!! Cheers

Hello all, I (original poster) am following up to say that we did happen to derive this cell line from the gentically engineered mosue model of melanoma. The methodology is described in the supplementray section of our latest report http://www.gastrojournal.org/article/S0016-5085(18)30411-6/fulltext

Dark-Field microscopy may assist with identifying positive cell but it may be difficult to identify where the cells are specifically in the tissue.

Regards

Chris

Thank you Sarah for your kind suggestions.

Thank you all for your assistance .