- Gary Lee Gilmore added an answer:5Hello everybody,does anyone have an explanation for the loss of surface expression(by FACS) after thawing?
Primary human stem cells expressing c-kit (also known as CD117) were obtained by cell sorting (99% purity ,using CD117 antibody),seeded,expanded and frozen(10%DMSO,90% FCS) at confluence.
Surprisingly,after thawing these cells don't expresse c-kit(DC117) evaluated by FACS analysis.
Does anyone have an explanation for that ?
I work with human UCB stem cells [CD34+/CD133+] that are also CD117+ [c-Kit]. I often use frozen mononuclear cells [post-Ficoll] and have never had any problem seeing CD17 on the surface of the CD34+ CD133+ cells. That being said, you are dealing with an adherent cell population. c-Kit is a receptor tyrosine kinase, like c-fms [CD115] and I know from studying mouse c-fms that freezing of an adherent c-fms+ cell line [RAW 264.7] results in loss of c-fms. Since RAW 264.7 is a transformed line, it eventually recovers c-fms expression after culture for a while. I do not know if your cell population will behave in a similar fashion or notFollowing
- 3Cell BiologyI am very new in cancer Biology. I want discussion related to critical mechanism of cancer progression.
The best way for you to have a good and comprehensive understanding of cancer cell biology is to read the review by Hanahan and Weinberg published in Cell in 2011.
- Scott Wong added an answer:6Can anyone recommend a good way to reduce lipofuscin autofluorescence in adult rat brains for later fluorescence in situ hybridization?
I am interested in quantifying transcription of several receptors in 7 month old rats whose brains have been perfused with PBS and PFA. The brains were then fixed for 2 hours in 4% PFA, cryoprotected, and sectioned on a cryostat. To label the sections I'm using Stellaris probes. The problem is I'm getting a background signal (small dots around the nucleus) in all my sections, including negative controls. This signal shows up in FITC, texas red, and Cye 5 filters, which makes me think it is lipofuscin. I've heard that Sudan Black can help with this but I haven't found anything on combining it with in situ hybridization. Does anyone have experience with this or any other useful protocols? Thanks.
Thanks for the advice everyone. I have a bunch of tissue sections available so I can try all of these and see what works.Following
- Tommy Lu added an answer:15Multiple compensation for individual flow panels?
I designed 5 panels of antigens to exam on flow-cytometry while each panel contains exactly the same fluorochromes. I noticed there are bleed-throughs in some of my channels even with compensation set up in advance.
The fluorochromes are as following: V450, FITC, APC, PE, eFluor 780.
The compensation is set up with both compensation beads and cells, both stained with single antibody. I choose either beads or cells for compensation setup based on brightness of the peaks.
Since I still have bleed-though after compensation, I wonder if it is necessary to set up compensations for each individual panel. Does antigens have anything to do with this? Or is it just my compensation going horribly wrong?
Massive thanks in advance.
My PMTV setting is similar to your first example with constant setting across each panel.
- 3Anyone have experience with the Inhibition of 26S proteasome?I am working on the Inhibition of 26S Proteasome in 26S proteasome enriched extracts from PC3 prostate cancer cells. I have done the experiment twice but there is a lot of difference in the inhibition pattern between the experiments with my compound. Is this anything to do with the extraction procedure or an experimental error?
The 26S proteasome extraction protocol includes suspending the cells in the lysis buffer, removing cell debris, ultracentrifugation at 3,00,000g and collecting the pellet which is enriched with 26S proteasome
May be the method detailed in the attached article could be of help for you.
- Kay-Dietrich Wagner added an answer:8Has anyone performed in situ hybridisation on adult mouse liver cryo sections?
I am trying to visualise the expression of a number of genes (including Arg1, Nr1d2, Per1 and Per2) in the liver (sections cut at 18 microns). However, I do not get any staining.
I know the protocol/solutions works because I use it routinely for brain sections and whole mounts. (My stringency washes after hybridisation step are: 5X SSC wash and 3 0.2X SSC washes). The probes work because I have used them in the brain.
I have tried reducing the hybridisation step to 40C and soaking the tissue in methanol overnight.
I am currently testing an increase in proteinase K digestion.
If anyone has any suggestions or advice I would be so grateful. Many thanks
Go down to 10um at least. Best results I obtained with the DAKO Genepoint kit and protocol.Following
- Antonio Diez-Juan added an answer:3Is it possible to knockout DNMT1 from human epithelial cells?
We would like to try to knockout DNMT1 in our lab for some experiments. However we are realizing that this might be lethal for the cells. I think there is only one report of DNMT1 knockout in differentiated cells (HCT116), but there is a production of a truncated DNMT1 protein instead. We would like to try it in kidney cells. I am wondering if anyone has ever try it and could give me some feedback, suggestions or opinions. Thanks!
I know is a bit dirty but to just move on and if you just need some invitro data to move on meanwhile you design a genetic tool, it could be useful to use some inhibitors that are not too dirty and widely used like VIDAZA (5-AZA) and 5-DACOGEN (5-DAC). Triggers proteosome-mediated DNMT1 degradation. There are others that inhibitors that do nott rely on DNA incorporation. http://www.jbc.org/content/early/2013/05/13/jbc.M112.443895.full.pdfFollowing
- Uwe Borgmeyer added an answer:4Database for Promoters and Transcription Factors?
Do you know a DataBase in which I can find promoter sequences of genes and the transcription factors that can bind them?? (i need to perform a CHIP assay)
Mattew's link is very helpful. I would suggest to try "other useful links", especially MEME.Following
- Alejandro Martin added an answer:2Clone having repetitive sequence insert and its discripancy upon scale up?
I have cloned my 3.3Kb insert having repetitive sequence in yeast expression vector, by directional cloning in E.coli. Positive Clone of 8.7Kb was confirmed by restriction analysis. While the same positive E.coli transformants when grown in 100ml LB, the restriction pattern observed is different. But the the digestion pattern is as expected when it is isolated from 5ml culture. Could anyone please let me know why this discrepancy observed from 5ml to 100ml scale up.
The more doublings of your population, the higher the probability that mutants bearing deletions will arise and overtake the culture. Most likely you already have mutants in the 5 mL culture, but at proportions low enough to pass below the limit of detection of your assay (restriction digestion + gel electrophoresis).
Have you tried E. coli strains designed to propagate repetitive sequences, such as SURE or STBL3?Following
- Daniel M Cohen added an answer:2Is it strictly necessary the chloramphenicol in LB agar plates for pLysS cells, or the ampicillin is enough?
If I add chloramphenicol only in the overnight liquid culture, is ok?
should the chloramphenicol be present also during the induction of the protein?
I agree with Domenico. Since the pLysS plasmid encoded chloramphenicol resistance, inclusion of chloramphenicol will maintain selective pressure, without impeding transgene expression. Nonetheless, you can safely omit chloramphenicol if you are transforming this strain with an expression plasmid, since the rate of plasmid curing (spontaneous pLysS plasmid loss) is quite low. Note that if you were trying to propagate the strain for other purposes (for example, making competent cells), keeping selective pressure on would be recommended.Following
- Ruth Bower added an answer:3Does anyone have experience with ATP assays on very small (5-10mg) tissue samples?
We have previously been using an all in one luciferase based assay, however validation of this has proved challenging in our application.
Any suggestions or recommendations welcome, thanks.
- Christian Winkler added an answer:6Hi there, can someone recommend a protocol or a test-kit to purify food samples with a high fat content to analyze fat soluble vitamins (Vit A)?
I want to analyze Vitamin A in very fatty samples (liquids as milk or paste-like food). The fat compounds seem to disturb my measurements. I am looking for easy to use sample preparations kits or a short protocol that leads to a high recovery of the vitamin.
Thank you very much !!!
I will try! Thank you !!!Following
- Nefeli Kakava added an answer:6Is there any reason not to mix two AAVs into the same injection? Ie is there a danger of some interaction?
I mixed AAV9 and some borrowed AAV2 (both with EF1a, FLEX and ChR2, but with 2 different fluorophores) into an epi so that I can inject them together, to assess effect of serotype directly at the same injection site (in Cre mouse).
I made the epi one week ago and kept in the fridge ever since, unsure if there is any danger of some sort of interaction! More generally, can one simply mix the viruses like that and still assume them to be independent? Do they compete in the brain to infect the same cells? Would be great is someone could share their experience/insights/refs! Obviously one could jsut do two separate injections to avoid this issue, but it would be easier, faster, and make for a better comparison if I just combine into one injection.. cheers
I agree with the rest!
I just wanted to add that it is more desirable to co-inject the same serotype of 2 different constructs, in order to raise the propability of hitting the same cells and the same amount.
Also, AAV9 has been reported to travel retrogradely (e.g. http://www.ncbi.nlm.nih.gov/pubmed/23600720) so you might expect to see fluorescence in other places in the brain, as well.
Hope it goes well!Following
- Meenakshi Khare added an answer:6Did any body have experience working with frozen blood for flow cytometry?
I am interested in some validation study which will need that...
Thanks everybody for your suggestions. WE are interested in doing lymphocyte phenotying, NK activity and intracellular cytokine staining. What we are interested in is that we will be getting whole blood samples from patients around the world and it is difficult some to get the blood within 24 h as that is what is required so we were thinking if we can still preserve integrity by freezing them and then isolating leukocytes .As samples will be coming from doctors clinic we cannot ask them to isolate cells and then freeze them...... we will be doing some comparision study but looking for if anybody else has an experience.........Following
- Rajeev Dwivedi, Ph.D. added an answer:77If you were a member of an academic committee, then how will you evaluate a Ph.D thesis of an applicant ?I wonder why a Ph.D Thesis from a reputed institute should have all the fame. I have seen some cases in which Dr. 'X' Ph.D from MIT or Cambridge or IIT have great chances to get selected in an Academic institute / Research centers.
Is this because of the Brand name or the quality of work?
If it is based on the quality of work then how will you assess it?
If it is assessed based on number of publication in reputed journals / patents and the impact towards scientific world etc.
if these requirements are there in a thesis from a low end University then will you accept that candidate to work in a reputed institute?
If yes, then what is the relation between a University and Ph.D it produce (Apart from providing infrastructures) ?
Certainly the Ph.D. from premium universities across the world get advantage. However, i have come across the Ph.D. from non recognized institutions and they are making great impact being a extra ordinary researchers. Premium institute researches get better access and better guidance but without novelty and without great publications no one sustain for longer period of time.Following
- Sian M Henson added an answer:13Does anyone have simple methods for telomerase activity?
Is there anyone to help me to find exact and simple procedure for telomerase activity for cell line characterisation.
Out of choice I would use the kit, the protocol is fine but it is a bit of a faff, but if you need to get results it will work fine. The only thing to take care with is being very clean when preparing the reagent other than that it's pretty similar to the kit. Real difference is it lacks the internal standard but you can spike it in if you're fussed. We compared the relative intensities and reported that.
Have a look at the following J Immunol paper:
we had to revert back to the old protocol as the kit was once again unavailable.
- Jochen Wilhelm added an answer:8Should you always log-transform qPCR and WB data (fold change data) before doing a statistical test?
I have some data-sets causing me and my limited statistical knowledge problems.
When handling qPCR values or quantifications of WB, you always "normalise" your values to a endogenous control / loading control i.e. a house-keeping gene. And then afterwards often to a control treatment as well.
However, this means the data is now fold change compared to a control. So all values less than control is between 0 and 1, and all values higher than the control is 1 to infinite. This heavily skews the data. To my understanding a parametric test like a simple t-test or ANOVA cannot be used, as it will it would handle fold change values 0.1 (A) and 1.9 (B) as absolute values, being equally apart from 1 (C), even though A is actually ten times less than C, while B is only 1.9 more than C.
I have a dataset from three replicate experiments, where I have transfected with either control siRNA or 3 other siRNAs (1-3) (4 samples).
I have subjected each replicate experiment to WB one at a time. In the WB I have blotted for 3 different proteins and a loading control (4 proteins). Afterwards I have quantified the WBs.
I.e 3 biological replicates, with 4 treatments, having 4 readouts (normalised to the same loading control).
In Prism, I have done two-way ANOVA on the values normalised to loading control, where I compared each treatment to the control treatment, using Bonferroni correction.
I have tried to do the test on both log transformed values and not log transformed data. I would guess it would be most appropriate to do the test on the transformed data.
However, the treatment with siRNA 3 results in small increases of some of the proteins with little variation between experiments, while another siRNA 1 results in decreased values but with big variation. When I do the ANOVA test on non log-transformed values the treatment with siRNA 3 is significant for these proteins, which seems right looking at the values and CI for both log-transformed and not transformed data. But when I do the ANOVA test on the log-transformed data it is no longer significant, while treatment with siRNA 1 (which had high variation), suddenly becomes much more significant. This does not seem right to me.
So my questions is should you always log-transform qPCR and WB data (fold change data) before doing a statiscal test? Or is their some problems in transforming your data, that will result in e.g. false negatives?
"However, it surprises me that it is so difficult to find any clear guidelines for doing statistics on WB and qPCR, as these are two of the most used methods of measuring expression levels in cell biology."
This is because many (most) biologists are not very literate in data analysis and statistics. They try to avoid it, and they usually switch off their brains as soon as they hear words like "logarithm". So there are many "lab-habits" growing, not all of them being very sensible. But they get published, and others take over the published procedures, without being able to judge the adequacy and efficiency of these methods. Don't get me wrong, I am not blaming biologists - it's not their fault. The problem is caused by an extremely bad education, and this is in major parts a systemic problem and not easy to solve.
" I guess I will have to dwell deeper into the world of statistics if I get the time. "
Take this time! It is important! I think it is better to have data from few experiments but a good understanding of the data (what is its relevant information content? how is it to be interpreted in the context of the research? etc.) than being busy in doing experiments, producing lots of data, but having no (solid) idea what this data actually tells.Following
- Sohini Roy added an answer:11Which media do you recommend for growing vascular smooth muscle cells?
I am used to growing human primary bronchial smooth muscle cells in DMEM - high glucose medium + 10% FCS, which works very well. Now I am planning to start growing primary human vascular smooth muscle cells (e.g. coronary artery), but I am uncertain if I can use the same medium or need to switch to commercial smooth muscle cell medium . Does anybody have information on this? Thank you in advance.
I just started working with Human Aortic Smooth Muscle Cells from ATCC. I am culturing them in DMEM (with 10%FCS, HEPES and Antibiotic/Antimycotic soln). They seem to grow very well. I want to know till which passage is it good to run my experiments with HASMC ?Following
- Prasanta Kumar Ray added an answer:3What are the morphological characteristics of fast growing Rhizobium japonicum?
I need to know the morphological feature of fast growing Rhizobium spp. For example, time required to form single colony, colony size, color, salt tolerance, BTB test feature, congured test etc.
Search literature and do the experiment yourself.You will know how it can be done.Following
- Stephan P Tenbaum added an answer:15Databases on receptors expressed by different cell lines?Is there any database where I can check what kind of surface receptors are expressed in different cell lines? Interested to compare two cell lines A431 and HEKn to see if they can be identified by receptors that are expressed on one but not on the other. If there is a source with all listed receptors for different cell lines would be nice to hear about it. Thank you.
I found this one
- Estanis Navarro added an answer:1Is there any one has murine μ and γ1 pre-switch sterile transcripts, and the post-switch γ1 circle transcript sequences?
We at least need I promoter sequences if there is no full length of transcript sequences available.
The promoter for mu-sterile transcripts is the IgH Emu intronic enhancer. You can read the following:
Nature. 1985 Dec 5-11;318(6045):475-8.
C mu-containing transcripts initiate heterogeneously within the IgH enhancer region and contain a novel 5'-nontranslatable exon.
Lennon GG, Perry RP
Hope this helps
- 2How many mouse cells are there in a xenograft tumor mass?I am trying to estimate the percentage of mouse cells in xenograft tumor samples. Does anyone have the estimation? What are the cell identities? Or any recommendation of paper or review about that?
You have to go back in the 1960s and 1970s literature with Mortimer Mendelshon (see two attached articles).
He "provided" you with more than precise responses to your question in bthe Oncology TextBook from V.T. DeVita (edition n°5 or 6) which must be available in your library.
f not, tell it to me and I will try to find back this book chapter.
- Christophe Praud added an answer:3How can I best determine extracellular (media) glucose concentration of differentiated C2C12 cells (non-radiometric)?
I'm trying to measure the extracellular glucose of my cells to get a feel for their rate of glucose uptake (from a fixed concentration of glucose), however I'm not really an experimentalist and am struggling to find a protocol for this. Our lab is equipped with spectrophotometers and all major enzymes and co-factors of the glycolytic pathway.
You can measure the uptake of 2 deoxy glucose by spectrophotometry using available kits.Following
- Natalia Duque-Wilckens added an answer:99+GFP fluorescence after fixationDoes anybody have experience with GFP fluorescence preservation following aldehyde or other kind of fixation? In our hands formaldehyde fixation (from freshly hydrolyzed 4% paraformaldehyde), rapidly quenches fluorescence.
What about acrolein?Following
- Nancy Turky asked a question:OpenWhat is the concentration of thrombospondin-1 protein in the serum of acute myocardial infarction patients compared to controls?
Please if you have an answer I kindly ask you to send me the related article
Thank you so muchFollowing
- Nii Sama added an answer:6Which method is good for periplasmic lysis of Escherichia coli?
which methods are good to disrupt the E.coli periplasmic space to extract protein and what is the mechanism of each method?
Thanks in advance.
Thank you so much dear Digvijay.Following
- Lalita Sharma added an answer:2What is the difference between LNCaP cells and LNCap FGC cells. Can we use LNCaP FGC cells in place of LNCaP cells?
I have to use LNCaP cells for Methylation specific PCR, but LNcaP cells are not available but LNCaP FGC cells are available. So Can I use these cells in place of LNCaP cells?
Thank u so much sirFollowing
- Lalita Sharma added an answer:4Can somebody please upload the photo of DU145 cells ?
We have Two types of DU145 cells in our lab and I am badly confused which one to use. So, Please upload some photos of DU145 so that I can come out of the confusion.
yes . plz sendFollowing
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