Tumor Biology - Science topic

Dear Colleague,

I hope this message finds you well. Creating a stable knockdown cancer cell line to be used for establishing a tumor-bearing mouse model involves several key steps, from designing and delivering shRNA constructs to selecting and validating the stable knockdown. Below is a detailed protocol to guide you through the process:

Steps to Create a Stable Knockdown Cancer Cell Line

Example Protocol:

By following these steps, you can effectively create a stable knockdown cancer cell line and use it to establish a tumor-bearing mouse model for your research.

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

Check out this protocol list; it might provide additional insights for resolving the issue.

Hello guys, thank you for your advice. However, it turns out that whole SU-8 wafer was made in the wrong way. There were missing another nanolayer of the certain chemical substance that makes it easy to remove PDMS from wells. In the first case, the residues of PDMS left in the holes. So now they adjust it and it is working well.

Dear sir Dr

Helper CD4+ T cells

Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate or assist the immune response.📷en.m.wikipedia.org › wiki › T_cell

T cell - Wikipedia

Hi Michael, here is a paper may answer your question.

Adoptive T cell therapy promotes the emergence of genomically

altered tumor escape variants

The main problem with freezing is ice crystal formation - the DMSO added to freezing medium helps to prevent that. It acts as a cryoprotectant. Without the DMSO, chances are good your cells will be damaged as ice crystals form both outside the cells and inside. If you're looking at whole tumors, by guess is that you'll have some live cells left in the center of the tumor, but experience significant cell death in the outer layer of tissue.

If you only need to store overnight, why not just store them in a refrigerator? It'll keep them cold, but not freeze them to death, and thus hopefully leave you with more cells to work with.

Hey Elisabeth Peer ,

I'd recommend doing a search on BenchSci and review published data for your best option (https://landing.benchsci.com/academic)

Please see attached for more info.

I hope this helps!

Maurice

Steve Mcclellan Hi Steve, Could you send me your old protocol for isolating viable cells after tumor digestion using collagenase/dnase.

Thanks a lot

We do HepG2 xenografts commercially (http://altogenlabs.com/xenograft-models/liver-cancer-xenograft/hepg2-xenograft-model/) and our standard procedure is to initiate treatment regimens when tumor volumes are around 100 mm^3. If your antibody is effective, then the growth curves will be substantially different, giving you good results. We use matrigel with one million cells, and that has worked remarkably well.

I agree with Roman about the volume of the injected mixture (100 microliters is a good starting point). Our xenografts (http://altogenlabs.com/xenograft-models/) are done with matrigel for the most part, and by the end of the study the growth curves are so clear that the initial volume of matrigel becomes insubstantial. As long as your growth curves have a relatively similar starting point, you'll have statistically significant results from an effective drug regimen.

I have seen in my experiment that the shape and cytoplasmic projection of human cells had changed when the sterilized broth media of 24 h cultured pathogenic and non pathogenic bacteria were added.

Dear Sir,

You can try with the expression level of cell surface marker such as CD44⁺/24^-. Also, you can try with EMT markers. 

In my work, I am using these CD44⁺/24^- marker. Cells having these markers are very aggressive and metastatic. (aggressive for triple negative and non-triple negative too)

Regards,

Utsav

Thank you!

The search for a spontanous mela cell line (not ex vivo gen. modified) is not that easy ^^

We used Bleomycin as the positive control for EAC (Ehrlich Ascites Carcinoma) cells.

Look in our paper-

Cheers 

You can digest tissue in RPMI-1640 (2-3% serum) containing 0.8mg/ml Dispase and 0.2mg/ml Collagenase P (Roche). Tubes should be incubated at 37˚C in a water bath or incubator and gently invert at 5-10 min intervals to ensure the

contents are well-mixed. After 30 min,gently collect the cells in another tube and add the fresh media with collagenase and dispase. Total process will take around 60 min. Centrifuge the cells and plate them in fresh media.

I would go with option number 1 from your literature, selecting for CD14 or CD11b or CD68 positive cells. By gradient centrifugation you would still have a lot of other cell types coming along.

I think we can use SRB assay for tumor spheroids too.

Ashleigh,

Thanks for the comments. I, too, am amazed by publications with this model. How can they 'realistically' and 'accurately' measure such tumors with large holes in them?  

From my experience with mouse MDSC and TAMs.

I think MDSC are well defined by markers as CD11b+GR1+ or CD11b+Ly6Chi. There are others makers in the literature but among all of them, I think these represent a good putative choice. Besides, TAMs, are a bit more complicated to get them. Surely, the marker CD11b overlaps between both populations but there are more specific markers from the alternatively (M2) activated macrophages that are not linked to MDSC. An example are the following; CD163, Arg1, Ym1 or Fizz1. 

Hi Mun,

I am currently culturing 4T1 cells, a murine mammary carcinoma line that displays both mesenchymal and epithelial characteristics. The media I have been using with much success is RPMI based, supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 μM L-glutamine. I have been subculturing at roughly 70% confluency in a T-25 flask and seeding the cells at 250,000 cells/flask.  

Hope this helps!

Chun-Mei Zhao,#1 Yoku Hayakawa,#2 Yosuke Kodama,1 Sureshkumar Muthupalani,3 Christoph B. Westphalen,2,4 Gøran T. Andersen,1,5 Arnar Flatberg,1 Helene Johannessen,1 Richard A. Friedman,6 Bernhard W. Renz,2 Arne K. Sandvik,1,7Vidar Beisvag,1 Hiroyuki Tomita,8 Akira Hara,8 Michael Quante,9 Zhishan Li,10 Michael D. Gershon,10 Kazuhiro Kaneko,11 James G. Fox,3 Timothy C. Wang,2,† and Duan Chen1,†

 

Go to:

Abstract

The nervous system plays an important role in the regulation of epithelial homeostasis and has also been postulated to play a role in tumorigenesis. We provide evidence that proper innervation is critical at all stages of gastric tumorigenesis. In three separate mouse models of gastric cancer, surgical or pharmacological denervation of the stomach (bilateral or unilateral truncal vagotomy, or local injection of botulinum toxin type A) markedly reduced tumor incidence and progression, but only in the denervated portion of the stomach. Vagotomy or botulinum toxin type A treatment also enhanced the therapeutic effects of systemic chemotherapy and prolonged survival. Denervation-induced suppression of tumorigenesis was associated with inhibition of Wnt signaling and suppression of stem cell expansion. In gastric organoid cultures, neurons stimulated growth in a Wnt-mediated fashion through cholinergic signaling. Furthermore, pharmacological inhibition or genetic knockout of the muscarinic acetylcholine M3 receptor suppressed gastric tumorigenesis. In gastric cancer patients, tumor stage correlated with neural density and activated Wnt signaling, whereas vagotomy reduced the risk of gastric cancer. Together, our findings suggest that vagal innervation contributes to gastric tumorigenesis via M3 receptor–mediated Wnt signaling in the stem cells, and that denervation might represent a feasible strategy for the control of gastric cancer.

If you want to achieve it a little bit easier, you apply  e. g. the point counting method according to E.R. Weibel, but also for this method the sampling procedure is essential (see morphometry / stereologyof the lung).

thanks, dear Sajid for your practical advices.

BM will give you macrophages.  M-MDSCs are found in low numbers in the spleen and other organs.  We were never able to get enough to study bio-distribution.  To get around this problem we labeled total spleen cells and used flow to identify who went where.  However, still not enough M-MDSCs to track.  We found you needed about 10e7 labeled cells to track over time.  The use of CSFE lets you follow cell proliferation as well. 

Dear AbdulMajid,.

That's my paper, we published it Alhamdolliliah.

Best 

Mannan 

Some articles that can help you.

Best regards

Robert

Hi Matthew,

You can use NOD/SCID mice. The macrophage function is not normal but not absent as well. It's just interrupted or delayed therefore you can try this well established animal model for xenografts.

All the best

Hey Siegel,

You can use this:

Starting material: Tissues stored as chucks under liquid nitrogen.

During handling the material is kept on ice. Before generating a lysate, the tissue is first cut into ~1mm cubes by using a razor blade on a glass plate held on ice.

The small cubes are then transferred into a hand held potter homogenizer with three ml ice-cold RIPA buffer per gram of tissue.

 Tough tissues like Prostate, Skin and Thyroid need incubated in the RIPA for 20

min on ice before starting homogenizing

 Medium tough tissues like Colon, Duodenum, Heart, Kidney, Skeletal Muscle or Tonsil, are kept in RIPA on ice for 10min before homogenizing

 Homogenize by pushing the piston slowly into the mix by continuously twisting the wrist thus turning the piston around its axe.

 Make sure all tissue chunks slide between the piston and the glass wall while homogenizing.

 Once the piston reaches the bottom, reverse the handling

 Keep the tissue submerged in the ice during the process

 Repeat the cycle until the tissue is liquefied

 Divide the liquefied tissues over 1.5ml tubes and centrifuge at 13,000rpm for 3 min at 4C

 Transfer the clear supernatant in new clearly labelled tubes. Take 20ul out for protein determination

 Protein determination is carried out using the BioRad Protein Determination Kit.

 Adjust the lysate to 5mg/ml by adding ice cold RIPA buffer

 Store in liquid nitrogen.

RIPA buffer: 20mM Tris-HCL pH7.4, 150mM Na\Cl, 1mM EDTA, 1% Triton-X100, 1%

sodium deoxycholate, 0.1% SDS with freshly added PMSF to 1mM and with freshly

added aprotinin and leupeptin to 5ug/ml just before use.

All the best.

Thank you very much for your answer.

I think, it  will help us a lot in our diagnostic.

1. "Extradural" is a descriptor for lesions in the spinal canal. This is outside the spinal canal, in the vertebral body.

2. Likelihood odds and Bayesian analysis (of which I am a fan) work only that far; at some point one needs to "fish or cut bait" decisionally speaking: I.e. Do something with the probability numbers. That something, in this case, is probably a biopsy or do nothing.

3. I'd say the lesion is not a hemangioma. A subtle fracture or infection are possible. A primary tumor or the vertebra is less than 1%. A metastasis is possible but not likely .( hystory and presence or absence of lesions at other sites (bone scan) could help. Lymphoma is a distinct an important possibility because of prognostic and treatment implications.

4. This is ONLY one image, of one of the imaging studies, of a whole patient with history, clinical context, etc.; very little information for a decision.

I'd be curious what the physician ordering the MR advised. If you want to talk about it, call me in Skype or WhatsApp

Andrei Vermont

Thanks Rainer! Yes, i found someone in Spain willing to collaborate on this, so all good. But thanks anyway for your kind reply!

The short answer is no, they can't, given that they will generate a very strong immune response in the recipient. Even in immunocompromised (IC) patients the time required for few blood cancer cells to give rise to a full-blown cancer is typically very long, so only a very severe and long lasting IC could lead to cancer. In this context however, the chances of getting cancer simply because of the severe IC will be much higher than those of getting it due to blood donations. In the SCANDAT (Scandinavian Donations and Transfusions) database there was no evidence of increase in cancer risk among recipients of blood from "precancerous" donors i.e. from donors that later on were discovered to have cancer. It is also true however that cancer can be transmitted after transplant of solid organs, which suggests that the global number of 'transplanted' cancer cells and the subsequent immunosuppression therapy required to avoid organ rejection are also a critical factors.

nf639 dreived from mammary tumor of transgenic female mice of MMTV Model carrying the ERB oncogene.  I  worked once with the FVB Tg mice which are suitable for your desire experiment. 

this is the stock number from Jackson labo. 

002376

i hope im helping 

good luck 

Here is an anti-granulin antibody mentioned:

Other literature:

http://www.eurekalert.org/pub_releases/2009-10/plos-pgh100609.php (currently offline)

Regards,

Joachim

A metastatic process is a 3D process occuring in an amazingly complex tumor microenvironment.

Any cell migration / invasion assays relating to "pure cancer cells forced to evolve in 2D dimension and prisoners of closed plastic dishes" have nothing to do with the actual 3D journey of metastatic cancer cells.

Here attached are various in vitro approaches that recapitulate some of the important aspects of an actual metastatic process.

Best regards

PS: I also added 3 reviews for those of you who would be interested by the metastatic journey. These are the first three articles here attached.

Essentially they are different epithelium, even grossly appearing so.

In the pancreatico biliary system, there are at least 4 distinct epitheium:

Our characterization of them morphologically seems simplistic at best, and one could expect additional division in the future.

Yeaton P, Kiss R, et al. Multiparameter digital image analysis of biliary, ampullary and pancreatic adenocarcinomas. Modern Pathol 1995;8:843-7

It remains an old story, as yet untold

Hi, Isaias. Using a caliper (vernier) is the fastest and cheepest way. To incease the precision, you could measure the three dimensions of the tumor and use formula V=0.5236(LWT) to calculate the volume (http://www.ncbi.nlm.nih.gov/pubmed/24682747). You could also wieght the tumors to assess their density. Computerized MRI would be the most precise method, but it is expensive and labor-consuming. 

 I would go for multi-modal MRI, mostly diffusion and T2 weighted imaging if it is active surveillance.

Thanks a lot Paul, I'll come back to you when I would have time to retry it!

oh great answers .... :) thank u

The CDTN and UFMG, Belo Horizonte -Brazil (Centro de Desenvolvimento da Tecnologia Nuclear and Universidade Federal de Minas Gerais) has animal PET  ,and humans PET-CT , you may wish contacting them       http://estatico.cnpq.br/programas/inct/_apresentacao/inct_medicina_molecular.html

Best regards,

Priscila Santana

I agree with Pierre.  For the most part, cancer is systemic and not the result of a localized exposure.  Exposures (diet, smoking, toxicants, etc.) that play a role in cancer initiation can cause both local and systemic epigenetic changes that influence the microenvironment. These exposures also cause inflammation.  Even in cancers that do not stem from inflammatory disease, inflammation is almost always present. However, if you are creating a local exposure and getting cancer both in that quadrant and in another, then, in addition to inflammation and inflammatory cytokines, there may also be epigenetic changes.  Everything in the body is connected, so the epigenetic changes aren't necessarily going to stay within the boundaries of the quadrant you have designated.

My paper coted second one above... will give you insight regarding many protocols.. if you still need help let me know.

Regards

Macrophages in the tumor tissue is generally considered to undergo "education" not to engulf tumor cells but to cause chronic inflammation to promote metastatic migratory behavior of tumor cells. Dormant cancer cells (G0 phase) are likely to evade from immunity system by erasing the surface MHC molecule. 

Thank you very much. I greatly appreciate your help.

Hi Camille, 

I always used the RIPA buffer with 1% Triton X-100 in the 1X lysis buffer. Both Brij and Triton X-100 are non-ionic detergent, thus I suggest you to add your compound at a lower concentration. 1% is suggested as final concentration in most of the lysis buffer recipes. To increase the ionic strength, add 1% sodium deoxycholate (final) in your lysis buffer. I don't think is a matter of the tissue you're working with. Usually, mouse melanoma tumors (e.g. from B16 cells) smashing easily just pipetting. Good luck!    

 Since the tumor is growing in 30-40% of your mice, I suggest that you extract the growing tumor from one mouse, expand cells in lab for few days, then reinoculate in large number of mice. also you can use these cells for next inoculation instead of the original cell line. I did that and it works

Thank you very much.

Hi Papiya,

actually I was even a little bit more cautious, and reduced it by 10%, 5%, 2,5%, 1% and then 0%. I have never tried a "normal" media without any FBS for a longer time, so I changed simultanously the media to one of the "serum-free"-media. 

Actually, if you need a lot of cell culture SN, you could first slowly change "your" medium to a serum-free media, first with the normal amount of FBS, I changed from 25%-50%-75%-100% always with 24 h between  Serum-free Media (always controlling the cell morphology; and always with FBS). Then reducing the FBS gradually.

If you don't need so much SN, just reduce the FBS gradually (always controlling cell morphology and cell attachment) and after a "short time" without FBS (I would recommend not more than 12 h, I have used 6 h) harvest the SN.

Carsten

Hello

Read this article ( Methods for Determining Spontaneous Mutation Rates ) by Patricia L. Foster , Where describes the proper design of a fluctuation assay, several of the methods used to calculate mutation rates, and ways to evaluate the results statistically.

Guo,

Normal beta cells contain glucose transporter 2 (Glut-2) and Glucokinase (GK). Both of these proteins have high Km (in the range of 13 - 15mM). Insulinomas tend to have Glut-1 and hexokinase, both are low Km versions. Transport of glucose into normal beta cells followed by its phosphorylation to Glucose-6-phosphate (the first step in glucose metabolism) is therefore dependent on the concentration of glucose in physiological range, whereas in the case of Glut-1 and hexokinase in tumor cells, even at the fasting glucose levels (3.5-5.5mM), the activity of these proteins is at their maximum level. Glucose metabolism, ultimately generates signals that are used by the cell to secrete insulin. This is why the insulinoma cells are always pumping out insulin at their maximum or near maximum levels even at 2 or 3mM glucose, which results in hypoglycemia. 

The B16F10 melanoma sub-cell line (from the B16 model) is usually employed to set up "pseudometastatic models".

If you inject the B16F10 cells into the tail vein, you will have the development of marked lunh "pseudometastases" (see the attached article by Mathieu et al., 2007).

If you inject the B16F10 cells into the carotid, you will have the development of brain mets (see the second attached article).

For subcutaneous model, you should graft the cells "on" the flank and not "into", otherwise you will have an i.p. model that will rapidly "destroy" the mice.

You can also use the B16 model for s.c. grafting but it is necrosing a lot (see Darro et al., 2005, here attached).

You should contact my colleague Véronique Mathieu, MD, PhD at vemathie@ulb.ac.be (she is unfortunately not registered on RG ...). She can help you with "some important details".

Best regards

Robert

Dear Riva,

Here attached you have three articles about the LLC model.

One article was "already" published in 1974 in the excellent journal Cancer Rwsearch.

A second attachd article shows a nice study using this model and published in PlosOne, which is a good journal.

A third article is a recent review and shows that the LLC model is often use.

There are ~4,000 publications with the LLC model in the PubMed database ...

I would not be too anxious with the LLC model ...

Best regards

Robert

Hello,

I do not have experience with work with tumor cells. However, I have almost 30 years of experience doing rodent surgeries. With respect to recovery of the rats or mice, the neurochemical environment, neuronal cell types, and glial cell types, there is a large degree of variation from one brain region to the next. For example some regions of the brain have proliferative zones and others do not. If one were to hope for refined answers to their questions, it seems like a very good idea to track the precise location/region into which you injected each time. There will be far more precision using stereotaxic injections. The Nature paper you mentioned - spoke globally of brain and other organs (the CNS is very diverse relative to other organs - so treating it with more precision is the norm and will likely yield better results).

So, it really depends on how precise an answer you need and how reliable you want your data to be.

Thanks a lot! But my main issue is the thickness of my samples, they are 80 microns thick. I am looking for more information, but thanks for your help anyway!

Katrin-

I have used the MIltenyi pan T cell kit.  It is easy to use, gives great purity (>98%, in my hands).  Miltenyi has streamlined the kit in the past year or so and it is now even easier and quicker to use. 

If you don't have access to a magnet, you could also try differential attachment.  T cells generally don't adhere to tissue culture plastic, but tumor cells, fibroblasts, other stromal cells will.  You can plate the single cell suspension and let the cells settle and adhere for several hours.  Then gently gently gently wash the wells with warm media or PBS and collect the non-adherent cells, the majority of which should be T cells.  The purity will be lower than the magnet, of course, but depending on what you plan to sequence (TCR?) may be good enough.

Here is a paper describing a live cell imaging system that was developed by some of my coworkers. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826880/   Our lab also provides foundry services to customize such devices. http://www.vanderbilt.edu/mfc/

With microfluidic devices, you are able to image your cellular system in a controlled environment that few other platforms are able to achieve. 

I realise this is an old thread - I'm having trouble getting hold of anti-mouse PD-1 (clone G4 ideally), would any one have access to this (and be willing to ship to Belfast, expenses paid!) or recommend a commercial alternative?

Thanks in advance!

sorry no

The short answer is YES. The real question is to what extent? See the relevant info in the link below.

Link: http://www.jimmunol.org/cgi/content/meeting_abstract/188/1_MeetingAbstracts/46.5

Ok thanks

Thanks for your rapid answers! I think I will try to initiate spheroid growth directly in the wells now. Hope that works, because every cell line behaves totally different in 3D-culture

Thank you, Fiona!

If your main assay is histological analysis then do not freeze at all but fix in formalin. Frozen sections are rarely as good as sections from formalin fixed tissues when it comes to preservation of tissue morphology.

If you still want to freeze, put tissue first in tissue freezing medium, then freeze. A more detailed discussion on this topic can be found here on Researchgate, foe example:

https://www.researchgate.net/post/Cryoprotection_with_sucrose_30_after_neutral-buffered_formalin_NBF_fixation/1.

There is no universal fixation/tissue preservation method. It depends on what your plans are, what you want to study, what downstream applications you plan to do.

Many antibodies for IHC work on formalin-fixed paraffin-embedded tissues (FFPE), many do not. you can get decent protein for Western blotting from FFPE sections but snap-frozen tissue is better. Same is true especially for DNA and RNA.

 Not sure how well DNA and RNA preserve after PFA/formalin fixation and freezing. But if the ratio of tissue to fixative and the size of the tissue are in a certain range, fixation should be fast and nucleic acid degradation at an acceptable level.

Thank you Mr. Tsyrlov. 

It also can be possible that your overexpression model was not transfected in a stable way.

Dear Stefania,

Whenever you are searching for expression levels whether basal or differential expression, please give it a try to the EBI Expression Atlas http://www-test.ebi.ac.uk/gxa/  As you can see, when searching for expression of both genes in "mammary gland cell line", it gives you the expression levels of both genes by specific cell line. Please see the attached image with the screenshot containing the cell lines/expression levels and number of experiments. Please let me know if cannot figure out how to get the results and I can send you a brief tutorial

Good luck!

Hi! This is an excellent question, and as you can judge by the two different responses you've obtained so far, this is not an easy question.

As mentioned above, these two mutations are classically considered to be exclusive and not occurring in the same cell. The data presented by Anastasia above, I will leave uncommented as I do not know of such dual-mutated cells in my experience. I do however know that there are clinical tumors where both BRAF-wt/BRAF-mut/NRAS-wt/NRAS-mut cells co-exist in the tumor. As also mentioned in the previous reponses, the BRAF-wt/NRAS-mut cells are not targeted by BRAF inhibitors, such as Vemurafenib. There are some case studies published, one in NEJM if I'm not misstaking, where they found an aquired NRAS mutation in the metastatic daughter tumor, where the primary tumor was "diagnosed" as BRAF-mutant and NRAS-wt and hence treated with BRAF inhibitors. In NRAS-mut/BRAF-wt I would expect an enhanced NRAS-related signalling.

Best of luck!

Concerning the use of matrigel with ovarian cancer, i rather suggest to use it for orthotopic or subcutaneous injections, as you already wrote, and not for the intraperitonel ones. Normally, in the abdomen, you expect not to form metastases in a shot time but ascites and the injection whith PBS or media, in my experience, is the best. For methastases is always better iv injection though in ovarian cancer case doesn't really make sense.

Good luck

I have worked with these cells and noticed that using trypsin to remove them from plates for passaging will cause damage after too many uses- I ended up having cells that looked very similar to the ones in the top picture. Therefore, manual scraping for long term/ multiple passaging has worked better for me, with less cell death.

Dear Hem,

Tumour measurements are best done with vernier caliper either in live animals or in excised tumours from sacrificed animals. Quantification is usually done by measuring length and width of the tumour and then using the ellipsoid formula to calculate tumour volume. Quantifying tumour size using pictures can only be reliable if all the pictures are taken using the same photographic parameters like distance of the subject from the camera, magnification etc. Even then, the orientation of the excised tumour could vary, and lead to error in calculations. Hope this helps.

Not really, it depends on your agent and its solubility properties (polarity). This will determine which dissolution vehicle you will use (ethanol,  acetone, DMSO, etc.) It is important to prepare a stock solution so you can keep the concentration of the dissolution vehicle below toxic levels.

What does it look like with only phalloidin staining? Are the out side cells stained then?  In which order do you stain? Or does it happen each way?

Dear Olivia

Thank you so much for sharing your experiences. I am thinking about a third option as well, It seems that Pluriselect products can be a good choice. Did you have any experiences with these non-magnetic beads?

 If you have only the functional domain, you may want to check whether this domain is responsible for binding or interacting with other proteins or DNA structure. And this can be assessed by a number of strategies such as yeast hybrid assays. I would like to suggest you to read this article in case you have not checked it yet. And also you may find important info about the structure and functions of this tumor suppressor in this article. Best of luck.

Dear Melanie, I would not recommend to spend your time. With frozen cells you can isolate DNA, RNA, proteins, to measure enzymatic activity, but you will never see T cell responses. If you intend to save viable (not frozen) T cells overnight, this is possible. The best way to save T cells by this mean would be placing into tightly closed flasks in complete medium and saving them in the dark at RT.

Hi Marcela.

My experience is with prostate cancer xenografts in NSG mice (we find these much better for xenos and PDXs) and they generally run for between 6 weeks and 4 months depending on how aggressive the cell line. I don't know CAL51 but I understand that our breast cancer colleagues run fairly similar length experiments for MCF7 cells. Also, we would never grow to 1000mm3 as that would be much too big for most UK project licenses. We usually go for 100mm3 (so about 4x4x5mm)

Alastair

Hi,

You can inject 4T1 cells from 5x10³ to 1x10⁵ in the up or low fat pad with volume 30ul to 50ul in Balb/c mice.  Then you can have lung metastasis and larger spleen ( 3-4 time weight (g) than normal mouse''s spleen.

Oi Elaine,

Eu sou Natássia, nós somos co-autoras no artigo do Guilherme Carneiro, ex aluno do professor Lucas. :-)

Nós estamos tendo resultados semelhantes, mas no nosso caso estamos observando outro efeito celular que é a inibição da diferenciação de células-tronco.

Concordo com o pessoal acima que a análise de Probit ou usar o valor máximo inibitório seria a opção ideal.

Mas no meu caso, por exemplo, a análise do Probit seria muito subjetivo e o valor máximo não serve para nossos objetivos. 

Uma opção que nos deram seria, no programa estatístico, estabelecer a maior citotoxicidade como zero, utilizando a ferramenta de constrain no GraphPad Prism. Lá, na função de Hill, você consegue colocar o bottom como 0, e ele calcula o IC50.

Como tb não conheço seus dados, não sei se seria apropriado.

Espero que tenha entendido! Se não, pode me escrever :-)

Abraços

Natássia