Glioma - Science topic

Hi Shaktiprasad, I am the Product Manager of the neural team at STEMCELL Technologies. We need a little more information and context for your question. Please contact our Product and Scientific Support team at techsupport@stemcell.com and a team member will troubleshoot with you.

Kind regards, Nusrat

Actually, I think you can add all the growth factors into your complete medium and store at 4 ℃. But the first thing you must take notice is that you should check the storage condition (the product data sheet) of all the growth factors. As far as I know, when storing at 4 ℃ refrigerator, EGF, FGF and PDGFRA are all OK before you running out the culture medium.

Joao Martin Martins, Gert Fricker ...Thank you!

The survAUC R package provides a number of ways to compare models link: https://stats.stackexchange.com/questions/181634/how-to-compare-predictive-power-of-survival-models

Shweta Singh Fantastic. Thank you very much. We are asking (neuro)psychologists, neurosurgeons, speech-language therapists, occupational therapists, physiotherapists, and psychotherapists to participate. If you have a few contacts you would like to share, please message me privately. We will be happy to reach out to these people directly, if it helps save your time. I am also providing my email address: MPolczynska@mendet.ucla.edu just in case. Best wishes, Monika

Hi Rodrigo.

I‘m having the same problem in my lab. Did you have some answer? I’m looking for someone in Brazil that is working with this model. Can I contact you?

Best regards,

I haven't worked with glioma cell before, but Lipofectamine can be toxic to some cell types. When I use Lipofectamine 2000 to transfect HUVECs (primary endothelial cells), I have to replace the transfection media after 4 hours to prevent cell death. I do not see any appreciable decreases in transfection efficiency (remains >90%) from doing this.

Hi Daniele:

There are a few things with DAPI nuclear staining:

- The DAPI has a long emission tail which cross talks with other channels

- There is a possibility of UV photo conversion of DAPI to "undergo UV-induced photoconversion to form green-emitting fluorophores" which might be having emission properties in the detection range (https://biotium.com/support/tech-tips/tech-tip-avoiding-artifacts-from-uv-photoconversion-of-dapi-and-hoechst/)

- Imaging DAPI signal last might solve your problem or use some of the NIR nuclear dyes whose emission is red-shifted away from your other signals

Good luck!

@Sathya Srinivasan

hi

you can used from PC3 & MDA-MB-231( good expression in mRNA and protein level )

good luck

The first pic. Shows encephalomalacia or a site of previous surgery, normally u see glioma hypointense in T1wi, but sometimes a hyper cellular thick capsule appears hyperintense

It is still under question (to my experience it not hereditary), however., recently it gained great attention by the researchers, see for example :

I suggest you follow : https://www.cancerquest.org/cancer-biology/cancer-development

Hello,

You may get the low-grade gliomas like oligodendroglioma, low-grade astrocytoma, gemistocytic astrocytoma from the adult neurosurgery ward near you. Every week there are such operations. You`ll get a mass of cells. If pediatric cells are more readily availables, ask for pilocytic astrocytoma. You will get it for sure!

Check this new database: https://lndb.grand-challenge.org/Home/

Yes, you could select male mice.

In general, male mice are selected for making a mouse glioma model (subcutaneous or intracranial). You can see this paper “ Huang, J., Jiang, G., Song, Q. et al. Lipoprotein-biomimetic nanostructure enables efficient targeting delivery of siRNA to Ras-activated glioblastoma cells viamacropinocytosis. Nat Commun 8, 15144 (2017) doi:10.1038/ncomms15144 ”.

In addition, due to the cyclical changes in estrogen in female mice, the glioma model of female mice may have larger individual differences.

Alyaa M Abdel-Haleem Mohamed if you are able to get NCI-60 cell lines panel?

I frequently perform intracranial injections with GL261 cells using 6 week old female C57BL/6 mice. After some optimizations of the cell number, i now inject 25000 cells in a volume of 2µl into the striatum; 2mm left of the sagittal suture and 0,5 mm anterior to the bregma at a depth of 3 mm from the dura, using a 2.5 µl Hamilton syringe equipped with an unbeveled 33G needle. With this setup i reach a 100% tumor uptake rate and an average of 26d before the onset of neurological tumor symptoms. I usually split the cells 2 days before the tumor implantation and seed them in a way that they, in the ideal case, exhibit a confluency of around 70% on the day of the implantation.Hope that information helps.

Hi Shen Wu:

i totally understand what you are talking since i had similar experience several years ago. Quite a few candidate experiments suitable for this aim, such as transwell assay, invasion assay, and wound healing assay, every experiment has its cons and pros, and none of them is edge-cuttingly convincing. Since this work is critical for my project, i tried all 3 of them, the results generally consistent, so i drew a conclusion.

If this piece of work is not so sedimentary for your whole project, it would be a good idea to choose the one Charles recommended above.

Best regards

Shiqiu

We also usually use laminin coated dishes when trying to transfer primary glioma cells to adherent, serum-free conditions, which we usually only do temporarily, e.g. for lentiviral transductions. We use the following procedure:

Laminin, L2020, Sigma Aldrich. Aliquot to 10-50 μl aliquots and store at -20C. Avoid freeze-thaw cycles.

The cells usually adhere perfectly within a few hours and can be kept without re-seeding for around 72h.

How many cells do you seed to a T75 flask? An important point i observed is, that you should not try to seed the cells in a too low density, so depending on cell numbers, you might try 10cm dishes.

to know if the tumor was induced you can label your cell ine with luciferase and then follow tumor growth and distribution using bioluminescence technic .

example: https://bio-protocol.org/e1762

Plz anyone help who are doing research in this domain

There are specific indications for surgery, radiotherapy and chemotherapy for both primary and secondary brain tumors. Histopathology, location, extent of resection, age, and multiple other variables play a role in decision-making for neurosurgeons and oncologists. Multiple guidelines help specialists, however, each patient is a different world.

Thank You!

Dear all,

Thank you again for your replies! I am having some trouble with the final risk model I am currently developing. I shall briefly explain how my study stands now.

I am trying to develop a risk model for venous thromboembolisms (VTE) in high grade glioma patients. 8.7% developed VTE. I have identified individual risk factors using descriptive statistics. Variables that have p<0.15 were included in the logistic regression. I have done collinearity diagnostics (no collinearity between variables was found) on included variables. I have subsequenty done logistic regression by enter method and included those variables with p<0.10 in my final model. I have calculated the weighted risk scores for each variable. Lastly, I have measured the AUC to assess model fit.

My questions concern the following:

1. I see that some papers include a risk adjustment score for the variables that have not been included in the final risk mode. I do not understand what this entails. This score is then used as a predictor variable in the logistic regression (this step i obviously missed).

2. From the literature I understand that bootstrapping is done when performing the logistic regression. However, I do not understand this concept and cannot find the criteria for using bootstrapping when performing the logistic regression analysis. I understand that the CI will more"reliable", but no more than that.

Kind regards,

Wills Floris filipe

Thank you so much Jennifer! One of my colleagues sent me a vial of frozen GL26 cells today, will grow them first and ask for help from the link you provided.

Thanks again!

Yanrong

I strongly recommend the following apoptosis detection kit.

Catalog number:  88-7003-42

URL https://www.thermofisher.com/order/catalog/product/88-7003-42

I'm sorry I have not tried to find out that percentage.

Most our studies about Glioblastoma use U87, and it's the most common cell line in current research. We also use U373, U251 at the same time to perform double verification.

I used to use this protocol. I used DMEM+10% FBS and passaged with trypsin. Other use DNAase/papain. For chamberslides, I think I used poly-D or poly-L-lysine coating followed by gelatin but bare tissue culture treated plastic worked basic astrocytes. You are probably actually isolating glial progenitors that mature into astrocytes in the dish. Also, there are irreversible changes when you expose to serum so defined medias (i.e. with DMEM + G2 or N2) also work and will give in vitro morphology closer to in vivo. Ben Barres paper from Neuron in January 2016 was pretty detailed for immunopanning to get purified populations as the cultures below will be mixed.

Day 1:

1. Put 10 ml DMEM/10% FCS in each 75 cm2 flask. Use one flask for every one or two pups (rat).

2. Dissect cortical hemispheres from P1-P4 pups.

3. Using a 10ml pipette, transfer dissected cortices to a 15 ml tube. (Use more tubes if necessary.) Rinse once with cold CMF.

4. Add 7ml CMF/0.25% Trypsin and strongly pipette up and down a few times with a 5 ml pipette.

5. Incubate tube in 37 water bath for 10 minutes, manually shaking tube every 2-3 minutes.

6. Add 7 ml DMEM/10% FCS to quench trypsin reaction. Pipette up and down.

7. Centrifuge suspension for 4-5 minutes at setting 3 on table top centrifuge.

8. Aspirate supernatant and re-suspend pellet in 2ml (more if needed) DMEM/10% FCS. Transfer to new 15 ml tube.

9. Using a mildly flame-constricted siliconized pipette (or 10ml and 1ml pipette), triturate ~15x. If several tubes were used, combine suspensions. Filter with a nylon cell strainer (only for preparation of astrocytes). For microglia use the entire suspension. Aliquot equal amounts to each flask.

Days 2-7:

1. At 24 hrs, give flasks a little shake and aspirate media. Replace with 10 ml DMEM/10% FCS. Shake again, aspirate, and replace media.

2. To obtain microglia, shake flasks for 2 hr. @ 160 rpm @ 37°C. Collect the media and spin @ setting 4 for 5 min. Then re-suspend microglia in a small volume of media and do a cell count. Dilute the microglia to the appropriate density and plate in 10% FCS/DMEM.

Confluency: Separating Astrocyte and Microglia Cultures

1. Put flasks into a plastic bag. Tape well with masking tape.

2. Dry shake 24-28 hrs at 37°C and at 250 rpm on orbital shaker.

3. Remove flasks from bag. Wash flasks in ethanol, or flame caps.

4. Remove media from flasks. (The media contains the microglia so save for microglial cultures). Wash twice with 5-10ml CMF.

5. Add 5ml CMF/0.25% Trypsin to each flask. Swirl, then incubate 8-10 minutes.

6. Bang flasks to loosen cells. Collect this suspension.

7. To the trypsinized cell suspension, add an equal volume of DMDM/10% FCS.

8. Centrifuge suspension for 4-5 minutes at setting 3 on table top centrifuge.

9. Aspirate supernatant and resuspend pellet in DMEM/10% FCS with mild trituration. Estimate cell number using hemacytometer.

10. Dilute astrocytes in DMDM/10% FCS and plate on polylysinized plates and/or dishes. Use for experiments within 7 days. You may want to switch to serum-free before experiments.

Hi Jane!

We are using the IN Cell Analyser, from GE. Our cells are U251 and A172 LentiLox transformed to express GFP, and we are capturing the images with fluorescence in intervals of ~2h. I do not think there is photoxicity in this interval, do not you? The CO2 and temperature are supposedly manteined in the same levels as in an incubator... I do not know if and how the equipment maintains the humidity level. I will try to inform myself about it...

I agree with my collegues. Many variables playing a role in radiation therapy for gliomas. In genral 60 Gy is the dose for high grade glioma in brain. But several ongoing studies on re-treatment and fractionate radation therapy.

Thanks ^Chanchai!

xCT inhibitor would exhibit the therapeutic effect. Glioma-induced edema would be attenuated by the SSZ. 

DLL1 is considered to be up-regulated in the compensatory manner instead of decreased expression of DLL4. Further, given the low affinity to the receptor, the sustained signal pathway is likely to require much more amount of DLL1 expression in your in vitro experiment.  

Hi Katrin,

sorry for this little bit of advertising. If you still looking for a protocol for FFPE of CD4 and CD8, I have the ones attached we use for our compan, unfortunately with "our" antibodies (we have recently produced 2 new antibodies especially for IHC-P in mouse tissue) .

Here are the links;

If you want to give them a try, I can offer you a free sample of both antibodies for testing. Please contact me.

Best regards,

Carsten

Hi John,

Thanks for you reply- I am slightly confused by your answer.

FeTPPS is a peroxynitrite scavenger so should cause a decrease in ROS production (we have results to show the decrease in ROS induced by FeTPPS as well as the decrease of apoptotic population when treated with FeTPPS). Therefore, we are under the impression that this decrease in ROS should cause a decrease in MOMP.

However, we are seeing a decrease in ROS yet an apparent increase in MOMP?

Is it possible that the FeTPPS as a ferric porphyrin complex my readings as it is read through the FL2 channel? Or another explanation?

I am not convinced that anoikis is a major way through which GBM cells die (see the Lefranc et al. attached article).

Validated protocols for cell death analyses are here attached (Galluzzi et al.).

You will encounter technical problems in trying to recapitulate actual anoikis in GBM cells because i) GBM cells rarely invade adjacent tissues (including brain parenchyma) through vessels and ii) they diffusely invade the brain parenchyma.

You can try to use an anti-integrin antibody in 2D GBM cell cultures to induce "mechanic" anoikis and then you should "simply" applied apoptosis-related protocols.

However, you should first determine what is/are the major extracellular components secreted by GBM cells during their "substratum attachment" on the plstic bottom of the flask (or even glass). The ECM secreted components vary from one GBM cell line to another one (see Belot et al.).

You could also use "anti-ion channel" compounds. For this point, I advice you to look carefully at the abundant publications by Harald Sontheimer on this subject.

A very nice article has also been recently posted by Luis Pardo on Kv10.1 with respect to GBM cells.

I hope it could help you.

Robet

It is very hard to have an established grade III glioma cell line because grade III glioma evolves towards grade IV, i.e. glioblastoma (GBM).

I think that the only way to have an actual grade III glioma in cell culture is to establish it from a patient with a proven grade III glioma.

Best regards

Robert

Taqman website clearly tells you what you expect in a SNP detection test. Go to Thermofisher website as taqman is under Thermo now. Look for Melt curves in a SNP detection test and you will have the datasheet. Its quite easy. Just that your Taqman primer should be in place. I see that they give ready made kits or you could get a primer probe designed around your mutation and use.

This link is for your mut R132H:

In the clinical settings, however, it is expected to be difficult to perform the transplantation of the precursor cells into the brain tumor tissue.TRPV1 activation inhibits EGFR-induced glioma cell proliferation mediated by the activation of Ca2+/calpain and resulting activation of protein tyrosine phosphatase 1B (PTP1B). 

Thank you so much.

Dear Reaearcher,

can read the following article to see if come with some information.

Int J Oncol. Author manuscript; available in PMC 2007 Dec 1.

Published in final edited form as:

Int J Oncol. 2006 Dec; 29(6): 1349–1357.

PMCID: PMC1661847

NIHMSID: NIHMS12617

SPARC-Induced Migration of Glioblastoma Cell Lines via uPA-uPAR Signaling and Activation of Small GTPase RhoA

2.    Suppression of the uPAR–uPA System Retards Angiogenesis, Invasion, and In Vivo Tumor Development in Pancreatic Cancer Cells

Bharathi Gorantla1,

Swapna Asuthkar1,

Jasti S. Rao1,2,

Jitendra Patel3, and

Christopher S. Gondi1

 

3.      Urokinase Plasminogen Activator, uPAR, MMP-2, and MMP-9 in the C6-Glioblastoma Rat Model

PATRICK J. SCHULER1⇓,

MARTIN BENDSZUS2,

SIGLINDE KUEHNEL3,

SVEN WAGNER4,

THOMAS K. HOFFMANN1,

ROLAND GOLDBRUNNER5 and

GILES H. VINCE3

 

J Cell Mol Med. 2008 Jan-Feb;12(1):174-86. doi: 10.1111/j.1582-4934.2008.00220.x. Epub 2007 Jan 9.

In vivo detection of c-Met expression in a rat C6 glioma model.

Towner RA1, Smith N, Doblas S, Tesiram Y, Garteiser P, Saunders D, Cranford R, Silasi-Mansat R, Herlea O, Ivanciu L, Wu D, Lupu F

J Neurochem. 1998 May; 70(5): 1819–1825.

PMCID: PMC2571951

NIHMSID: NIHMS73238

Evidence for κ- and μ-Opioid Receptor Expression in C6 Glioma Cells

Laura M. Bohn, Mariana M. Belcheva, and Carmine J. Coscia

E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, U.S.A.

Address correspondence and reprint requests to Dr. C. J. Coscia at Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104, U.S.A.

Author information ► Copyright and License information ►

Thanks,

PR Bollam, MD, MSChe

Hey David

I will definitely recommend MS-HRM if you want to assess the methylation level of the promoter region (or another smaller region of interest) for a lot of samples. It is very sensitive for detection of low-level methylation. The only requirements for the technique is a real-time thermocycler with optics capable of collecting fluorescent data points (LightCycler480, Roter-gene Q, 7500 Fast Real-Time PCR system and 7900HT Fast Real-Time PCR System.

There has already been published work about MS-HRM for DNA methylation assessment of the MGMT promotor region. Hence, you don't need to waste time on optimization of primers, annealing temperature, composition of master mix etc.

PMID:25859283: Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome

PMID:19483476: Primer design versus PCR bias in methylation independent PCR amplifications

Good luck!

Hi Pablo!

Thanks for the answer and the very valuable links. I go and check them out.

best,

Béla

Did you get this cell line? I have been searching this cell line lately.

Hey! For a kill curve you use untransfected cells, as you want to determine at what concentration non-resistant cells die. The concentration you are looking for is the lowest concentration that kills all cells after 7 days.

We have just used an antibody against HLA-A. This is one of the MHC class I proteins. Worked well.

Hi Bernhard,

we conducted several research approaches to this issue- from my clinical experience I meant metastases /recidives of high grade gliomas. On the other hand we have a bright clinical experience within the use of boswellia and therefore could observe it`s significant life-quality improvement in patients suffering of high grade gliomas.

Dear Gaëlle, 

Check these papers out:

Generation and validation of a prognostic score to predict outcome after re-irradiation of recurrent glioma.

Combs SE, Edler L, Rausch R, Welzel T, Wick W, Debus J.

Acta Oncol. 2013 Jan;52(1):147-52.

Results of nimotuzumab and vinorelbine, radiation and re-irradiation for diffuse pontine glioma in childhood.

Massimino M, Biassoni V, Miceli R, Schiavello E, Warmuth-Metz M, Modena P, Casanova M, Pecori E, Giangaspero F, Antonelli M, Buttarelli FR, Potepan P, Pollo B, Nunziata R, Spreafico F, Podda M, Anichini A, Clerici CA, Sardi I, De Cecco L, Bode U, Bach F, Gandola L.

J Neurooncol. 2014 Jun;118(2):305-12

Try NCCS in Pune

Dear Samira, 

I think attached paper may help you.

best

mehdi

Thank you to both of you!!

@Christopher

Thanks for the adding a great answer to this discussion. Although I most definitely agree that GFAP is not a good marker to differentiate tumour cells from normal brain cells, I'm a little surprised that only fewer than 5% of the samples you describe were positive for GFAP. There is vast literature describing GFAP immunopositivity having correlation with the WHO grading scheme of neuroglial tumours and indeed it is used regularly in the clinic as a tool in histopathological diagnosis. Yes, GFAP is not a high grade glioma biomarker, because low-grade gliomas, like diffuse astrocytomas (grade II) also express GFAP. Although by no means is this relationship universal, and GFAP immunopositivity can be varied in different glial tumours - weak, strong, diffuse etc., a complete absence of GFAP immunopositivity in a GBM would at least warrant an investigation into a differential diagnosis of a more primitive/embryonal entity, perhaps a PNET and I do not believe this is  the case for >95% of high grade glioma patients.

In order to make suggestions, it would be helpful to know what your research question is. There are many many models of gliomas that can represent different histologies, tumour grades, genetic backgrounds etc. Are you wanting to do orthotopic xenograft models, RCAS-tva, transgenic models...?

This is, of course, a complex question and the prior answers have only added levels of complexity.  Tumoro neuropathology is inevitably and properly going the way of hematopathology--molecular genetic (and epigenetic) information adds precision to prognostication and to choices of therapy and so becomes an essential part of the diagnosis; pretending otherwise (and staying with H&E alone) is just putting one's head in the sand.

That said, the molecular genetic data need to be developed with objectivity and with correlation to histological and immunohistological data.  In my opinion the conclusion that 1p/19q co-deletion is "diagnostic" of oligodendroglioma was jumped at too rapidly, and did not account for (1) classical oligodendroglioma by histology which are not co-deleted, in adults (rare but definitely occurs), (2) pediatric oligodendrogliomas which are usually not co-deleted; (3) the variably demonstrable presence of neuronal differentiation using ultrastructural or immunohistochemical markers of such differentiation in so-called oligodendrogliomas, with and without 1p/19q deletions; (4) the rare demonstration of 1p/19q co-deletion in tumors not histologically compatible with a diagnosis of oligodendroglioma.  When one considers the enormous spatial heterogeneity in any large resection specimen of diffuse gliomas, whether looking at histology, at immunostains as markers of protein expression, or at molecular genetic criteria, it becomes clear that looking at the histological diagnosis of OA and then taking one sample for genetic analysis and rejecting the mixed diagnosis because there is either 1p/19q co-deletion in that sample or because there is p53 mutation and ATRX mutation in that sample is inappropriate.

Histological appearances presumably result from an interaction with tumor genetic characteristics and environmental influences.  We understand little about the genes suppressed or activated in 1p/19q co-deleted gliomas, but the available studies point to abnormal regulation of genes controlling neuronal differentiation.  Taking the cancer stem cell hypothesis at face value here, OA is the resujlt of differential activation or suppression of neuronal differentiation factors in different cells or different parts of these tumors, under the control or influence of local environmental factors about which we know almost nothing.  So it is premature to think that the diagnosis of OA can be legislated out of existence.

My own diagnostic practices have evolved from how I understand the literature such that I now more frequently make a diagnosis of "glioblastoma with oligodendroglioma" to recognize a tumor which has histological components of oligodendroglioma or neurocytoma but which also has necrosis with pseudopalisades and florid vascular hyperplasia.  Similarly, I find I see more cases of "glioblastoma with PNET" in adults than I used to recognize.  By using panels of immunohistochemical markers I have for a long time recognized that neuronal differentiation is more common in all of these tumors than standard texts, or the WHO, concede.  Whether this is of clinical importance is not wholly clear, although some studies from the Paris group have shown that glioblastomas with neurofilament protein immunopositivity have a better prognosis compared to those that do not.

The important data from the past few years suggests that the histological diagnosis may be of less therapeutic ("predictive") and prognostic importance as compared to the genetic data; thus once one identifies a diffuse glioma, knowing that it has 1p/19q co-deletion, or IDH mutation, or ATRX mutation, appears to be more important than whether one calls it from histology astrocytoma, oligodendroglioma, parenchymal neurocytic tumor ("extraventricular neurocytoma"), or mixed glioma (OA).  For practical, clinical purposes, this information assumes paramount importance now, until we better understand the factors regulating glioma cell differentiation and whether that understanding will lead to better targeted therapies.

One last note--the WHO won't be coming out with a wholly new CNS tumor classification for some time.  The "interim" update from the Haarlem meeting was "allowed", as I understand what was said by leaders of that meeting, because the WHO was pressured by the neuro-oncology community to recognize the significant changes that have occurred since the 2008 publication of the last WHO "blue book", and this has happened also in the hematopathology world (in fact, more so).  However the WHO bureaucracy is apparently wedded to finishing the current series of blue books across all organ systems and cancer types before allowing work to begin on the next series, so a wholly new WHO CNS tumor book can't be expected until late in this decade, if then.  Don't hold your breath.

What is your solvent and what is the final concentration in the medium?

Hello Robert. Thank you for your answer. However to answer your question on why bEnd.3 is chosen, it is largely due to personal interest. It is then supported by the few articles especially by Brown et al. 2007 which claimed that bEnd.3 has rapid growth, maintains BBB characteristics over repeated passages, forms the functional barriers, and amenable to molecular interventions. 

Erlotinb is a lipophilic molecule with a secondary amine (logP 3.2 and pKa 4.6. according to chemicalize.org). The crystallization, or precipitation, you are describing is likely due to the very low solubility of erlotinib. The solubility is approximately 0.9 µM at neutral pH (estimated by Henderson-Hasselbalch relationship using a reported solubility of 0.4 mg/mL at pH 2 and the pKa of the compound).

While DMSO does provide an opportunity to introduce the compound to the assay it will not keep the drug in solution unless the DMSO concentration is very high. Depending on the robustness of your cells you could try to adjust pH or solubilize your compound using a surfactant or a cyclodextrin. It may however be a better idea to try to adjust concentrations used in the experiments as any solubilizing measure will affect the outcome. Due to the low pKa of the amine your medium needs to be quite acidic (i.e. pH 3 or 2) before a substantial increase in solubility could be expected. This would probably be very detrimental to the cells. Solubilizing efforts involving surfactants or inclusion complexing agents can potentially reduce the tendency for the drug to precipitate, but will also reduce the fraction of the drug that can interact with a potential target.

Best regards / Jonas Fagerberg

Hi, in the different culture media or other condition, cells will secret different enzyme (types, amount..). And EDTA cannot be used in this study, it will inhibit the metallo-.

Vinculin has a higher molecular weight (around 100-110 KDa) and it is often used as loading control in Western Blot, so you can try it.

Thank you all for your answers!

Anyway, any more details or informations will be always welcome!!

Thank you

Thank you Marianne for the Link, but Prof. N. Bechrakis doesn't have experience in tumor induced retinas, so i am still trying to find anyone who has done or any ideas about to investigate the invasion of tumor cell line  on retinas models.

Even mild and non-specific in vitro manipulations like cell transfection can elicit pro- differentiation signals that make cells to proceed with their developmental program. Professor Potter used to say that 'cancer was a blocked ontogeny' and this seems to be the case in your experiments with the glioma cell line. In fact, a mild solvent like DMSO was found to signal through MAP kinases and PKC and cause leukemia cell lines to differentiate. These unexpected findings highlight the importance of controls. Because you have good controls, you can conclude that the morphological differentiation you observe is due to the transfection procedure seen in the negative controls. 

Could your blocking agent be degraded? Some antibodies require and additional H2O2 application prior to applying to avoid a lack of specificity.

Hi Sara,

Thanks a bunch! I might have to take a look at a panel of cells. I am interested in GBM cells that are essentially "normal" for NF1 that also do not have proteasome mediated degradation. It looks like that figure should provide me with a good starting point. I suppose the main issue is to define "normal," which is a little difficult to do! 

 

Thanks again,

Robert

many thanks

I have made cell lines from patient glioma samples, and FACS sorted them once they were established cell lines, but I have not FACS sorted straight from tumor, so take these suggestions with a grain of salt:

- You're going to have a problem with a lot of necrotic tissue, so you could try taking separate portions of the tumor and hoping that some of them may be better than others.

- For disassociation of tumors or neurospheres I use Accumax (from Millipore). Never use trypsin, it will cause the cells to all clump together.

- After disassociation repeated washes (until you no longer have cloudy media/PBS after centrifugation) may help.

I worry that the debris may just be a fact of working with disassociated tumors, but the CD133 researchers seem to be able to FACS sort fresh tumors...

I would also be needing human normal astrocyte cell line as a control in my experiments. Let me know if any one of you could share it.

Everything on these markers including the sequences can be found here:

Nestin (NES) http://www.ncbi.nlm.nih.gov/gene/10763

OLIG2 http://www.ncbi.nlm.nih.gov/gene/10215

SOX2 http://www.ncbi.nlm.nih.gov/gene/6657

Good luck!

Thank you Dear Prof. Duntsch for your valuable feedback. I will try to incorporate your suggestions in my work.

Thank you once again.

With Regards,

Sai Manohar

Hi Andrei,

I have to agree with Emma that none of the "quenching" protocols really do a perfect job. I have in my own experience, however, found that the best tradeoff between getting rid of auflu and not completely killing you signal of interest is Sudan Black B. Its a mess, is not perfect but its better than the other methods I have tried. Try 0.1% solution (made with 70% ethanol). It has to be heated until it dissolves completely.

In general human H1, human and mouse U6 should all work in human, mouse and rat cells. Since shRNAs are highly expressed from these three pol III promoters, so it's hard to tell there is any tissue/cell specificity among these three promoters by knockdown efficacy, i.e. it may not matter much which one you choose to use. However H1 promoter is more flexible for nucleotides at transcription start.

This commentary may give some nice insights...

The exact method will depend on the drug and nanoparticle formulation but here is a method I used for polymeric nanoparticles:

To measure drug release from nanoparticles, nanoparticles should be lyophilized, weighed, and resuspended in buffer (I used two different PBS/0.1% Tween-80 buffers, one at pH 7.4 and one at pH 6.5, but you can use what ever buffer is suitable for your drug/nanoparticle preparation) and then incubate in a 37 °C water bath. At various time points (I measured between 15 min and ten days) an aliquot of eluted drug medium should be removed for quantification; this volume must replaced with fresh buffer to prevent sink conditions. Drug release can be quantified by measuring the absorbance of the release media using a plate reader or HPLC, this value, of course, will be unique to the drug you are using. As a control you should also measure blank nanoparticles (with no drug) and make suitable standard curves for your drug concentration in buffer.

Please see the two attached papers of mine for methods and if you have any other questions please let me know.