- Venkata Suresh Reddy Vajrala added an answer:2The best compositions of assay buffer and washing buffer for Renal Carcinoma exosome-ELISA ?
I just started working on Exosomes-ELISA research. I found out that people (who are working in this filed) are using different compositions of Assay and Washing buffers for their work.
The main objective of my request here is to know the right compositions of Assay and Washing buffers for capturing & detecting exosomes using antiCD9. Could you please share your recipes here on this board?. Any help regarding this would be greatly appreciated.
Thank you very much in advance.
Hello Marina ...
No I am not. I have decided to start with (PBS + 0.5%BSA + 0.05% Tween 20) and superblock buffer.
Thanks for your replyFollowing
- Lidia Mazur added an answer:22Is it necessary to starve the cells prior to any drug treatment?I want to check effect of my drug U0126 on cellular proliferation (cell line A498). How can I decide the time of incubation with drug and after how much time I should proceed with proliferation assay?
Do you want to obtain the effects of starvation or your drug on A498 cells ?
You have to determine the time of incubation of cells with the tested agent.
You can analyze the cell count/ cell viability and IC50 value using MTT assay,Following
- Dennis France added an answer:1Does anyone know of a mouse tumor cell line that has mutated Von Hippel Lindau (vHL) and/or that over-expresses HIF1-alpha?
I am working with an agent which I think would be particularly effective In tumors that overexpress hif1-alpha (like human renal cell carcinoma), but I am not aware of mouse lines which display this. (It is an immunotherapy experiment, so orthotopic human line in nude SCID mmice will not be appropriate.)
Bill Kaelin at Harvard would know where to direct you as h has a long-standing interest in this area.Following
- Anna Antonina Sobocińska added an answer:3Is anyone familiar with 786-O renal cell carcinoma cell characteristics?Is there anyone familiar with renal cell carcinoma cell lines, such as 786-O? I am trying to find what kind of cells they are, for example if they are proximal tubule epithelial cells. Also, would the culture conditions RPMI 1640/ 10% fetal bovine serum media be ideal? Any idea or reference would be very appreciated.
In my lab We use RPMI 1640 with 10% FBS and antibiotics. It works well - 786-O is not demanding cell line and it grows really fast - as well as for an example ACHN cell line. Please be aware of which cell type would you like to work on - clear cell, papillary and so on - sometimes they are with wrong description. If you have any more questions please do not hesitate to ask me. Have a nice day.Following
- Jordan Jahrling added an answer:3Can anyone suggest a good housekeeping for western blot analysis of normal kidney and renal cell carcinoma tissue homogenates?
We normally use beta Actin for normalizing RCC cell lysates. We tryed both beta actin and tubulin on tissue homogenates without any good results.
Can I assume you're using whole cell homogenates and did not fractionate (ie nuclear, cytosolic, membrane, etc)? If so you can also give GAPDH (35kDa) or Cofilin (20kDa) a try. If you have higher MW areas clean on your blots, you may try Vinculin (125kDa)Following
- Divaker Choubey added an answer:1How to encourage spontaneous immortalization of kidney cancer primary cells?
Is there a way to encourage spontaneous immortalization or proliferation of renal cell carcinoma primary cells? These are cancer epithelial cells. I have no problems growing the cells (DMEM supplemented with 10% FBS), but they grow slowly and undergo senescence after 4-5 passages.
One way to promote immortalization of the primary murine fibroblasts is to maintain cultures at an appropriate cell density. However, it is difficult to predict whether epithelial cell would respond similarly. Therefore, I would plate cells at higher cell density (as opposed to lower cell density) to assess the response of cells.Following
- Gregor Babaryka added an answer:3Does anyone know how to reliably differentiate between adrenal adenoma from metastatic RCC in adrenal gland?
IHC, EM or simple histology - everything is OK, but I need 100% of assurance.
RCC should be positive for the immunohistochemical markers cd10 and rcc, weakly positive for ckpan. Adrenal Adenoma should be positive für inhibin, possibly for melanA and weakly positive for synaptophysin. Both should be positive for vimentin...In deed, distinction can cause Problems, good question...Following
- Shruthi Kanthaje added an answer:8How can I dissolve Sorafenib tosylate?
Hi all, If anybody is using sorafenib (a multikinase inhibitor used as a chemotherapeutic agent in liver cancer, renal cell carcinoma etc.,) for in vitro studies? Literature suggests its solubility in DMSO is 200mg/ml. But in our lab it does not dissolve even at 1mg/ml (DMSO). It is not dissolving in water. If anybody has used it.. please let me know how you have worked with it?
Youtake oh... Thank you for the response...Which sorafenib do you use?Following
- Go J Yoshida added an answer:2TGF-β–induced EMT in renal cell carcinoma lines?
does anybody has got personal experience with TGF-β–induced EMT in renal cell carcinoma lines?
Thanks a lot
I think the following reference would be informative for you. The synergistic effect of TNF-alpha and TGF-beta ligand is considered to be important for tumor cells as well as pigmental epithelial cells in the retina to exhibit EMT.Following
- Joe Graymer added an answer:6What is the treatment options for metastatic non clear cell RCC with brain mets?
Brain mets and non clear cell histology were always the exclusion criteria for most of the recent clinical trials that approved targted therapy for RCC, and treatment of those patients is currently debatable. please find and share the way you mange this group of patients
Is this of any application in this case?
Anticancer Drugs. 2008 Apr;19(4):431-3. doi: 10.1097/CAD.0b013e3282f5d336.
Remarkable shrinkage of sarcomatoid renal cell carcinoma with single-agent
Fujiwara Y(1), Kiura K, Tabata M, Takigawa N, Hotta K, Umemura S, Omori M, Gemba
K, Ueoka H, Tanimoto M.
(1)Department of Hematology, Oncology and Respiratory Medicine, Okayama
University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
A 60-year-old Japanese man presented to our hospital with a painful left hip.
Computed tomography showed a tumor in the left kidney and metastases in the left
gluteus maximus muscle and lung. The pathological diagnosis of a biopsy specimen
obtained from a metastatic lesion in the left gluteus maximus muscle was
sarcomatoid renal cell carcinoma. On admission, his general condition was
extremely poor. He was confined to bed because of severe left hip pain and
confusion, possibly caused by hypercalcemia. This seriously ill patient suffering
from advanced sarcomatoid renal cell carcinoma was treated with single-agent
gemcitabine, resulting in symptom relief and a dramatic improvement in his
status; all of the tumors had regressed significantly by the 11th dose of
gemcitabine. These findings indicate that single-agent gemcitabine is one of the
few chemotherapeutic agents effective for palliation in patients with sarcomatoid
renal cell carcinoma, even those with poor performance status.
PMID: 18454054 [PubMed - indexed for MEDLINE]
Cancer Chemother Pharmacol. 2008 Feb;61(2):223-9. Epub 2007 Apr 19.
Pharmacokinetics of gemcitabine in tumor and non-tumor extracellular fluid of
brain: an in vivo assessment in rats employing intracerebral microdialysis.
Apparaju SK(1), Gudelsky GA, Desai PB.
(1)College of Pharmacy, University of Cincinnati Medical Center, Cincinnati, OH
PURPOSE: Gemcitabine is a pyrimidine nucleoside analogue anticancer agent that
has shown promising anti-tumor activity in several experimental models of brain
tumor. However, the pharmacokinetic behavior of gemcitabine in the central
nervous system, especially in brain tumors is currently not well understood. In
this study we evaluated the gemcitabine brain extracellular fluid (ECF) in normal
rats and in ECF obtained from tumor- and tumor-free regions of glioma-bearing
rats, to better understand the availability of the drug to brain and brain
METHODS: The brain ECF pharmacokinetics of gemcitabine were investigated
employing intracerebral microdialysis following intravenous administration of 10,
25 and 100 mg/kg doses in male Sprague-Dawley rats. In the second phase of the
study, gemcitabine (25 mg/kg) was intravenously administered in rats implanted
with C6 gliomas and ECF samples were simultaneously obtained from the tumor and
tumor-free regions of the brain. Serial blood samples were obtained for
evaluating the plasma pharmacokinetics of gemcitabine. Non-compartmental approach
was employed for the analyses of the brain ECF and plasma pharmacokinetics of
RESULTS: Following intravenous administration, gemcitabine rapidly distributed
into rat brain. At doses equivalent to 10, 25 and 100 mg/kg, the brain ECF
gemcitabine AUC (area under the plasma concentration--time curve measured over
the last sampling time point) values were 2.46 +/- 0.7, 3.20 +/- 1.1, and 9.06
+/- 3.0 microg h/ml, respectively. The brain ECF concentrations of gemcitabine
declined in parallel with plasma concentrations. At the three doses evaluated,
the relative brain distribution coefficient (AUC brainECF/AUC plasma) of
gemcitabine ranged from 0.07 to 0.09 suggesting limited gemcitabine availability
to brain tissues. Studies on C6 glioma-bearing rats revealed that following an
intravenous dose of 25 mg/kg, the AUC values in the tumor-free and tumor-brain
regions were 4.52 +/- 2.4, and 9.82 +/- 3.3 microg h/ml, respectively. Thus, the
AUC of gemcitabine in the tumor ECF was on average 2.2-fold greater than the
corresponding value in the tumor-free ECF of the brain. Plasma pharmacokinetics
of gemcitabine remained unaltered in tumor-bearing animals, when compared to
plasma pharmacokinetics in healthy animals.
CONCLUSIONS: Our findings suggest that the overall brain exposure to gemcitabine
is likely to be low as evident from the relative brain distribution coefficient
of <0.1. However, the exposure is likely to be considerably higher in the brain
tumor relative to tumor-free regions of the brain. The higher drug levels in
brain tumor compared to the non-tumor region may facilitate selectively higher
cytotoxicity against brain tumor cells.
PMID: 17443325 [PubMed - indexed for MEDLINE]
Regarding the general problems in brain metastasis, I always was in doubt about the so called: 'eloquent' metastasis, that do produce an specific neurological deficit, and thus, the suspicion exists that any local therapy would make the same lesion the metastasis induced, and end result will be the same, however, some 'eloquency' is needed for diagnosing a brain mts, you perform standard CNS imaging as part of work-up only for certain tumors, but: are there any series, if somebody attempted this, of 'eloquent' brain mts invasively treated?
- Mauricio Marti Brenes added an answer:3What methods are used to detect similarities between AML (angiomyolipoma) and RCC (Renal Cell Carcinoma) ?
Thank you for this information.
Las diferencias a nivel molecular y a nivel imagenologico, las que expone el articulo anterior y aun mas simple, se difencian muy bien en una tomografia axial computarizadasFollowing
- Amit Gupta added an answer:2How can I make a single cell suspension from renal cell carcinoma? Does a different tissue have a different protocol?
If you have the answers, I hope you also tell me the titles of articles your answers are from.
Try this one it might help you.
Here I am posting few suggestions given by one of RG member that will help you in protocol optimization.
I thing forming single cell suspension will vary....but I am sure you need to check it
- Maurizio Salvadori added an answer:3How common is native kidney renal cell carcinoma in a renal transplant patient with stable graft function for 26 years?
Have one such patient who underwent nephrectomy with stage 2 RCC. What should be the changes in immunosuppression in such scenario?
The frequency is higher than estimated. We should to check by US al least every two yearsFollowing
- John Samuel Banerji added an answer:4What is the best time for renal angioembolization (RAE): before nephrectomy in large hypervascular or locally advanced renal cell carcinoma?
The ideal timing of nephrectomy after embolization is unclear.
I don't think I would agree with Dr.Theodor Klots, with regards to the timing of RAE.
One week is too early-ideally it is done just a day before surgery-sometimes even hours before.You want to operate before tumor lysis syndrome kicks in. Also, the longer one waits, the more edema and destruction of surgical planes is there, especially at the hilum.
In fact, there are a few papers, where a balloon catheter is threaded unto the renal artery, in the OR suite, and then left in. After opening, the balloon is inflated, to occlude the arterial inflow, in larger tumors, before ligating the renal artery.
One of the major problems during nephrectomies for very large vascular tumors, is the "venous hypertension"-especially in the presence of tumor thrombus or large collaterals.
Using RAE just prior to surgery, in that setting is useful.Following
- Gamal Abdul Hamid added an answer:3What is the best treatment option for patients with ccRCC and CML?Would you combine imatinib with TKIs or mTORI?
I think there is no clear guidelines for the treatment of concurrent RCC and CML. The determination of the CML status as primary or secondary is important.
I think after nephrectomy the determination of suspected getetic mutation is important before begining Sunitinib, sorafenib or Beva.
The combination of two target therapies is effective , but what is mandatory to register the genetic mutation during treatment.Following
- Shomik Sengupta added an answer:3Can anyone help with an undifferentiated carcinoma of kidney?A 46 years old male patient with locally T3 renal cancer with huge interaortocaval lymphadenopathy and 5 cm parenchymal lung metastases and biopsy proven undifferentiated carcinoma has uncontrollable abdominal pain resistant to narcotics. Is a salvage surgery a feasible option before systemic treatment?
Cytoreduction or palliation are justifiable indications for nephrectomy in the presence of metastases. I presume this is undifferentiated adenocarcinoma of the kidney? These tend to have poorer response to systemic therapy - if the majority of the disease burden is in the kidney & lymph nodes, and they appear surgically resectable, I would definitely recommend surgery. It may be worth also exploring whether the lung lesion can also be resected perhaps thoracoscopically.Following
- Mary Khorami asked a question:OpenWhat is the colony morphology of renal cell carcinoma? How to diagnose them?Please share a picture of renal cell carcinoma colony before staining.Following
- Kathryn Parker added an answer:20How to avoid rapid pH fluctuation in DMEM medium?Before storing the DMEM (sigma high glucose) at 4°C. I set the pH 7.2, and next day I checked the pH of the medium. After warming up at 37°C the pH rose to 7.8. What could be the reason and will it effect the growth of my cell line? I am using ACHN and A498 (renal cell carcinoma cell line).Dear Gerald and Wolfgang,
Thank you Gerald for your great answer! It has certainly cleared up a few things: I will be sure to keep the caps on my media bottles tight, this was something I probably wasn't doing enough at some points.
So thanks to both of you, your answers have been very helpful. I am new to this forum but so far all my experiences with it have been very good, I think it is a big success!
Kind regards and many thanks again,
- Samer Salah added an answer:6What is the current practice in the absence of good evidence of cytoreductive nephrectomy in the current era of targeted therapy for metastatic RCC?Are you for or against performing cytoreductive nephrectomy for patients with metastatic clear cell- renal cell carcinoma who are treated with VEGF-TKI therapy !
In the absence of evidence, one would like to listen to the opinions and have an idea on the current practice. Any supporting evidences are highly appreciated.Dear Stephan Kiessig,
Thank you so much for the attached article. Interesting data ! However, my question is about patients with METASTATIC renal cell carcinoma who are treated with the novel targeted therapies, and the evidence that you provided suggests a benefit of immune therapy as an adjuvant therapeutic strategy following nephrectomy in patients with NON-METASTATIC renal cell carcinoma.
There is debate regarding the necessity of performing a cytoreductive nephrectomy in patients with metastatic renal cell carcinoma who are treated with VEGF-TKI therapy. The evidence supporting survival advantage for cytoreductive nephrectomy in the metastatic setting is evident only for patients treated with Interferone Alfa (based on two randomized trials).
In the absence of evidence supporting the benefit in patients treated with targeted therapy, it is wise to see the opinions and the current practice of experts in the field, and to see what is their opinion in that clinical setting and whether there are subgroups of patients (depending on the extent of metastasis, the bulk of the primary, the MSKCC risk category, or any other factors) that are likely to derive benefit. And this is the main reason why I added this question for discussion.
Please check the attached reference which discusses this controversial issue.Following
- Samer Salah added an answer:5What is the standard therapy for sarcomatoid renal cell carcinoma with brain metastasis?I have a 52 year old male patient who presented with transient dysarthria and numbness at the right side of the face. The patient was transferred to the emergency room where an MRI of his head showed two lesions; one in the left parietal lobe, and the other is located in the right cerebellar hemisphere. The size of the lesions is around 1.5 cm in maximum diameter each. Both lesions were highly suggestive of metastasis. The neurological symptoms resolved completely within few hours of his presentation. A CT scan of chest and abdomen showed renal mass arising from the upper pole of the right kidney and invading the peri-nephric fat, with possible invasion of psoas muscle, and an enlarged subcarinal LN (around 3 cm in maximum dimension). CT guided biopsy from the kidney mass was performed at our center and confirmed the diagnosis of sarcomatoid renal cell carcinoma (not sarcoma arising in the kidney. It is the sarcomatoid subtype of RCC).
Bronchoscopy and EBUS biopsy from the subcarinal LN was carried out and showed negative LN. Additionally, an MRI for the abdominal wall was not suggestive of psoas muscle invasion by the tumor as it showed clear separation of the psoas muscle and the tumor, and as such, the kidney mass is resectable according to the opinion of the urologist at our center. The patient has history of coronary artery disease, had previous catheterization and coronary stent two years ago, he is also a known case of hypertension. His hypertension and coronary artery disease are well controlled with medications, his left ventricular ejection fraction is ok, and he is now asymptomatic and has a peformance status of 0.
What is the best therapeutic option for this patient ?Dear Tamer,
Thank you for your suggestion about gemcitabine if chemotherapy is needed in the future. Any supportive evidence for that ?Following
- Ladislaus L. Torday asked a question:OpenWhat is your opinion? Should we stop TKI treatment after a metastatic renal cell cancer patient reached complete remission?Think about side effects and the risk of recurrence.. :)Following