Cell Biology

Cell Biology

  • David Greening added an answer:
    What is the best storage of conditioned media (at 4C/-20C/ -80C) for subsequent isolation of exosomes?

    I wish to isolate exosomes from MSC conditioned medium and would like to know what is the appropriate temperature for storing it in case i am not proceeding with exosome isolation immediately after conditioned media collection .

    1) Can conditioned media be stored at 4C/-20C for a week prior to exosome isolation. Will it damage the exosomes or degrade the biomolecular cargo present within them.? 

    2) I am aware of the fact that conditioned media can be stored at -80C , but somehow feel uncomfortable doing the same as i think thawing conditioned media from -80 to 4C might damage exosomes resulting in poor exosome yield .

    So i need help in two things

    1) what is the best temperature (4C/-20C) for short term storage (1-2 weeks) of conditioned media that would cause minimal exosomal damage ? 

    2) if storing at -80C , what is the best procedure for thawing conditioned media without damaging exosomes..

    Thank you

  • Imtiyaz Rather added an answer:
    How can I determine if a phenotype effect is Genetic, Epigenetic or ChoroEpigenetic?

    In a mammalian cell for a reason “A” – viral infection, environmental pressure, stress, etc., - occurs an asymmetric cell division (ACD) at M-phase for a factor “B” or a complex “C”. If, this factor or complex is present in one of the two daughter cells for next generations – the phenomenon could be a Genetic or Epigenetic event? Could be consider that the reason “A” affects genetic inheritance?

    Imtiyaz Rather · University of Phoenix

    Phenotypic effect is related to somatic origin while the genotypic e

  • Björn L.D.M. Brücher added an answer:
    What do you think about circulating nucleic acids damage DNA of healthy cells by integrating into their genomes.?

    Although I am not able to evaluate the methods I believe that this work just published (attached) offers the expected results, for new ways to look at the pathophysiology. If the results can be confirmed, everything will have to be reviewed in light of this germinal work, even if the amplitude of the concrete consequences is currently not determinable.

    Björn L.D.M. Brücher · Theodor Billroth Academy

    Please find below strong evidence supporting the new cancer hypothesis:

    Japanese scientists treat lung cancer patients with anti-inflammatory and –fibrotic atrial natruretic peptide and show that patients have by this lower recurrence rates

    Summary:
    Interesting approach from Japanese scientists: the authors published in 2011 a paper in which they could show, that circulating tumor cells in pulmonary veins during the manipulation of lung cancer surgery could be a prognostic indicator for early recurrence [Funaki et al. Eur J Cardiothorac Surg 2011;40(2):322–327]. Further this group showed, that ANP downregulates inflammatory response and having a prophylactic effect on postoperative complications due to lung surgery [Njiri et al. J Thorac Cardiovasc Surg 2012; 143(2): 488–494 Nojiri et al. Eur J Cardiothorac Surg 2013; 44(1):98–103; Eur J Cardiothorac Surg 2012; 41(6):1330–1334]. It is important to mention that ANP - besides an inhibition of the renin-angiotesin-aldosteron path through specific binding to the guanylyl cyclase-A (GC-A) receptor - has an anti-fibrotic effect (!) [Li et al. Curr Cardiol Rev 2001; 5(1):45–51 and Kishimoto et al. Curr Cardiol Rev 2009; 5(1):45–51]. Now the authors combined these findings and applicated ANP during curative lung cancer surgery and found that the recurrence rate (versus control) was lower.

    Nojiri T et al.: Atrial natriuretic peptide prevents cancer metastasis through vascularendothelial cells. Proc Natl Acad Sci U S A. 2015 Mar 16. pii: 201417273. [Epub ahead of print]

    http://www.pnas.org/content/112/13/4086.abstract.html?etoc

    Independent of the support of the recent published new cancer paradigm: 

    “Epistemology of the Origin of Cancer: a new paradigm"

    BMC Cancer 2014; 14:331: 1-8:

    http://www.biomedcentral.com/1471-2407/14/331


    and its its deeper explanations by 

    “Cell-Cell Communication in the Tumor Microenvironment, Carcinogenesis, and Anticancer Treatment"

    Cell Physiol Biochem 2014; 34: 213-243

    http://www.karger.com/Article/FullText/362978

    this could be a very useful approach for future peri-operative application in cancer surgery

    - and that is the reason why we are here for.

  • Rutger A.F. Gjaltema added an answer:
    Which method is the best for CRISPR/Cas9 assay?

    I will use Santacruz CRISPR knockout plasmid. I need a recommendation regarding company's transfection reagent and media (with or without FBS).

    Rutger A.F. Gjaltema · University of Groningen

    plasmids are plasmids, and efficiency depends mostly on cell types. I use lipofectamin LTX plus for both primary and cell lines with a wide range of plasmids (incl. CRISPR, not from santa cruz)

  • Taral R Lunavat added an answer:
    Can you recommend a commercial kit for proliferation assays?

    Can anyone tell me which could be the best kit commercially available for performing the Proliferation assay?

    Taral R Lunavat · University of Gothenburg

    Thank you everyone for your suggestions. In our lab we discuss this issues quite a lot and MTT/XTT these assay usually define the metabolism of the cells and not actual proliferation. Whereas on the other hand, BrDU incorporation gives us the right information when it incorporates into the DNA. THus when cells are actually proliferating, we come to know how much of it is proliferating compared to control.

  • Oscar Quintana Bustamante added an answer:
    In transient off-site targeted mutation transformations like the CRISPR/Cas-9, will genomic DNA be mutated and heritable without Cas-9 proteins?

    Transient gene transformation allows expression, right? So, if the Cas protein is expressed to do its job, and its job is to mutate this particular portion of the DNA, then would the mutated gene be inherited, and without the rest of the transiently expressed construct? 

    Oscar Quintana Bustamante · Ciemat-Centro Investigaciones Energéticas, Medioambientales Y Tecnológicas

    Yes, the dsb made by crispr/cas9 system will be inhereted by the daugther cells. Cas9 and guide can work transitorly, however their result will be durable in the genome

  • Dolores C Carrer added an answer:
    What is a good container for fixation in formaline?
    What is the best container in which to put a tissue sample to fix in 10% Neutral buffered formaldehyde? Can I use just a centrifuge tube or should I use a glass bottle?
    Dolores C Carrer · Instituto de Investigación Médica Mercedes y Martín Ferreyra

    thank you all!

  • Sunnie Wong added an answer:
    What is the best loading control for membrane fraction western blot?
    What is the best loading control can be used proteins in membrane fraction other than catenin proteins
    Sunnie Wong · Tulane University

    Maybe this will help?

    http://www.abcam.com/primary-antibodies/loading-control-guide

  • Alessia Vivanti added an answer:
    Mitomycin C precipitates in PBS solution after storing a -20 degrees. Is this deleterious to cells/compound?

    Hi everyone, I have recently purchased mitomycin C from SantaCruz to inactivate MEFs and since insufficient information was provided by the manufacturer on reconstitution and stability I had a read around and decided to dissolve the dry powder in PBS at a final concentration of 0.5mg/mL. I then prepared aliquots of 300uL and stored them at -20degrees protected from light. The following day after freezing I thawed a few vials to use and did not notice anything unusual (colour was nice light purple and no flocculation). However after 10 days I took some more vials out and noticed that an insoluble precipitate has formed. I did some research around and could not find anything exhaustive. Sigma says that once precipitate forms the solution becomes toxic/ineffective. However this paper (http://www.ncbi.nlm.nih.gov/pubmed/11821684) says that mitomycin in PBS retains stability up to one month when stored at -20 in PBS. I would like to ask in your experience, has this ever happened to you? Have you noticed any difference in efficacy? Many thanks in advance

    Alessia Vivanti · Stem Cell Medicine Ltd

    I dissolve it in water and store at -80. I never noticed any precipitation

  • James M Phang added an answer:
    What evidence is there that these is no enzyme Ornithine cyclodeaminase-like activity in mammals?

    Mu-crystallin (CRYM 16p13.11-p12.3.  in human)  is a lens structural protein in diurnal marsupials and an enzyme in mammalian possible involved in the potassium-ion recycling system; in addition it is a mammalian homologue of Agrobacterium ornithine cyclodeaminase..

    In mammalian forebrain ketimine reductase was identified as mu-crystallin. It was also seen that CRYM encoded "adenine dinucleotide-nicodinamide phosphate (NADPH) -regulated thyroid hormone-binding protein (THBP)"; this identifies a new role for thyroid hormones in regulating mammalian amino acid metabolism.

    There are many publications that correlate these enzyme activities to  various pathological events.

    Analysis of human tissues detected abundant expression of CRYM in heart, brain, skeletal muscle, and kidney, lower expression in lung and liver.

    James M Phang · National Institutes of Health

    Using radioisotopically labeled P5C, we  showed that proline inhibitsf P5C reductase activity.  However, we never studied P2C reductase.activity.

  • René Assenberg added an answer:
    How to grow Flp-In Trex 293 cells in suspension?
    I am wondering if there is any way to grow Flp-In Trex 293 cells (from Invitrogen) in suspension?

    Flp-In Trex 293 is adapted from HEK293 cell.
    René Assenberg · Novartis

    Hi,

    HEKs can be grown easily in suspension (I work with them and Expi293 cells all the time), you just need to adapt them through progressively diluting out your starting medium with new suspension medium (eg FreeStyle), or buy them straight from a vendor. 

     There`s plenty of protocols around to help you there – follow Ian`s suggestion there -  but this paper has some details on adapting Trex cells to suspension (not done it myself yet):

    http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0063679

  • Andras Szollosi added an answer:
    Can anyone help with an issue with site directed mutagenesis?
    I have been trying to incorporate two point mutations in a 8kb plasmid using the Quick Change II site directed mutagenesis from Stratagene. The two mutated sites are 8bp apart. Also, I used the Stratagene primer designing software to design the primers. So far I have been unsuccessful getting a band after PCR. Couple of times after DpnI digestion, I did bacterial transformations (even though there was no band in gel), however, did not see any colony. Moreover, using the same PCR set up, I see blue colonies on X-GAL, IPTG coated plates for the control plasmid that comes along with the kit. I pretty much follow the kit guideline to set up the PCR reaction mix. Also, I tried with different amt. of template (5ng-50ng) keeping the primer concentrations constant, but no luck yet! Bottom is the PCR set up I used. Any idea will be highly appreciated.

    Initial denaturation: 95C for 3min
    18 cycles of 95C for 50sec.
    55C for 50sec.
    68C for 18min.

    Final extension: 68C for 8 min.
    Hold:4C.

    I always run a gel taking 10ul. of the reaction mix after PCR and use the rest for DpnI digestion.
    Andras Szollosi · Semmelweis University

    You could also try to increase the amount of PCR mix used for transformation (5-10 ul) and use different types of competents (in our hands TOP10 supercompetent cells work best). I would not worry too much about the band in the gel I had successful transformations several times without seeing any gel band. Also be careful not to mix the antibiotics in the plate (vs resistance gene of your vector).

  • Holly Pondenis added an answer:
    What can I do to make LNCaP cells grow in a monolayer rather than in aggregates?
    My LNCaP cells started growing in clusters/aggregates (cells grow on top of each other) rather than in a monolayer. I can actually see small bumps/clusters by eye in my flask.
    Holly Pondenis · University of Illinois, Urbana-Champaign

    My LNCaP cells were clumped and not growing well.  I passed them thru a needle and syringe to get single cells as suggested and replated. They are finally growing well!

  • Pavel Trávník added an answer:
    How long does the effect of a-amanitin on RNA polymerase II last when it is added into cell culture medium?
    Any references are welcome
    Pavel Trávník · Sanatorium REPROMEDA

    Dear Gerelchimeg,
    in my opinion the alpha-amanitin is in vitro very stable molecule, therefore I expect, that the effect may persist for the whole culture duration. Long time culture with alpha-amanitin is often published, e.g.:
    Magdalan J, Ostrowska A, Piotrowska A, Gomułkiewicz A, Podhorska-Okołów M, Patrzałek D, Szelag A, Dziegiel P. Benzylpenicillin, acetylcysteine and silibinin as antidotes in human hepatocytes intoxicated with alpha-amanitin. Exp Toxicol Pathol. 2010 Jul;62(4):367-73. doi: 10.1016/j.etp.2009.05.003. Epub 2009 Jun 24.
    Best regards
    Pavel Travnik

  • Eric Brevers added an answer:
    How can I store human serum and plasma for long time?

    I have human serums and plasma, and I need to store them for long time, for example 2 years. Does anyone have experience with this task?

    Thank you. 

    Eric Brevers · Centre Hospitalier Universitaire de Liège

    Hello,

    u can store them at -80°C as said before but the time of conservation will depend of the components u want to analyze.

  • Motaher Hossain added an answer:
    Can anyone recommend a good and free tool/program for drawing biological diagrams?
    I need to construct bacterial invasion pathways in epithelial cells.
    Motaher Hossain · International Centre for Diarrhoeal Disease Research, Bangladesh

    Try these two

    http://www.cellillustrator.com/

    http://www.celldesigner.org/

  • Baishakhi Ghosh added an answer:
    Can a power failure during the first spin of PBMC isolation ruin the buffy coat layer?

    We have power trips going on quite frequently in our lab and to say the least this is a nightmare for my PBMC isolations. I was wondering if it would ruin the layer separation permanently or is there still a chance to get a nice layer by resuming the spin after electricity restoration? I wanted an opinion from someone who has experienced such an event.

    Baishakhi Ghosh · Chest Research Foundation

    Well the power cuts does have an impact in getting the layer separation. We had seen that we don't get the PBMC layer at 55-67% percoll gradient. Instead the PBMC get dispensed in the 55% percoll gradient. So we take the entire 55% Percoll suspension and make up the volume to 50 mL with 1X PBS followed by centrifugation, which does gives a good pellet of PBMC later.

    Hope this helps. Good luck!

  • Mark K Chee added an answer:
    Is there a scaffold (skeleton) composed of protein filaments inside mitochondria?

    I've read a lot of articles on cytoskeleton and mitochondria, but still haven't got a clear answer about existence of internal scaffold inside this organelle.

    Mark K Chee · Duke University

    Olivia, you've made a good point from a macro-scale. On the micro (molecular)-scale, from what I've read, I believe that the cristae's structure and shape are determined by the scaffolding or shaping proteins that underlie the inner membrane, which I believe was what Volkan alluded to.

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790458/

  • Gloria Bora Kim added an answer:
    Possible toxic effect of L-Lysine?
    Does anybody know if lysine (100 mM) added to the cell culture media has toxic effects?
    Gloria Bora Kim · Pennsylvania State University

    is p-l-lysine cytotoxic when used as a coating material and not washed with water or PBS after coating?

  • Sharada Kankonkar added an answer:
    Is there any kind of change in pH of cell while it is dying due to viral infection?

    Viruses are using cell machinery for its amplification. After  enough number of replication cycle it leaves cell and attack next cell. Is there any chance of changing pH of that infected cell during amplification process?  I mean whether pH values of cell is increasing at the time of  its genome replication and translation process and pH value decreases when cell is dying because of degrading enzymes. 

    Sharada Kankonkar · Kokilaben Dhirubhai Ambani Hospital & Medical Research Institute

    I agree with the concept dying cells change the PH (acidic) due to degrading of enzyme.

  • Christian Müller added an answer:
    What are proper housekeeping genes in the plasma membrane fraction for western blotting?

    I studied the regulation of Caveolin-1. I used coomassie staining of PVDF membrane as loading control for western blotting of membrane fraction (isolated by differentiational centrifugation). But reviewer is not convinced. He asked me to use proper housekeeping gene control because according to him it is not a reliable method. 
    Can you please suggest me whether beta-Actin be used in membrane fraction loading control? Some feel assertive, whereas some feel that  actin is a contamination in membrane fraction.For this ambiguity, I did not use it as a housekeeping gene in membrane fraction. I failed to find a exact answer in the literature resolving the issue of cytoskeleton proteins in plasma membrane fractionation.Can you suggest me any reference?

     Except cadherin, Na,/K-Atpase, what housekeeping gene can be used, since they are not housekeeping for my experiment? 

    Hoping your response.

    Regards,

    Christian Müller · Otto-von-Guericke-Universität Magdeburg

    NaK-ATPase (alpha-subunit)

    Pan-Cadherin

  • Alice Brosius added an answer:
    What are some good devices to effectively and quickly mince human adipose tissue?

    We routinely get between 50 and 150 grams of adipose tissue that we process for stromal vascular cells - we currently mince by hand with scissors, but it's a huge time drain and takes a long time, which may be affecting cell viability. Does anyone know of any devices to help in this process? We are considering anything and everything, from a kitchen blender to expensive tissue homogenizers.

    Alice Brosius · University of Michigan

    Carl, what kinds of tissue have you used the cheese grater on? This is one of the options we are considering.

  • Vasileios N. Georgakakos added an answer:
    What does modal number of a cell line mean?
    For HCT 116, the modal number at 45 is 62 %. What does it mean ??
  • Patrick Baril added an answer:
    Why are crystal violets measured at 590 nm or 570 nm?
    I have search through the internet and found that its maximum absorbance is at 590 nm. However, I have found many research to use 570 nm for measuring the de-stained crystal violet in ethyl alcohol.
    Patrick Baril · CNRS Orleans Campus

    Dear all,

    I fellow with interest your topics.

    May I ask for another point. Does it is possible to measure absorbance directly on plate with cell monolayers stained with crystal violet and dried ? I just start working with this dye for taking illustrative picture of cell-death induced by chemotherapeutic drugs. In the same time I would like quantifiy the assay. I saw that some people solubize the dye by treatment with SDS 1% but It is a long procedure and in our hands not accurate. Sometines genomic DNA is an issue to read the sample.

    Many thanks in advance for your comments experience with,

    Best regards,

    Patrick

  • Akhilesh Sharma added an answer:
    Is anyone familiar with High Reference flow cell binding in Biacore?

    I get very high reference Flow cell binding from 100-1000 RU(over injections!) I am struggling hard to get that down (<20 RU) I use 1X HBSP buffer! is this purely electrostatic? The conductivity of the MilliQ I use is ~2us any help?

    Akhilesh Sharma · Biocon

    OK, when i immobilise a receptor on the CM5 chip in one of the flow cells and leave the  other flow cell blank (after activation and deactivation with EDC and NHS) even before i start injecting analyte, I get high (>50RU) binding in the reference (blank) flow cell from the start up cycle where there is essentially no drug. Moreover, as and when the drug is injected in the subsequent cycles the binding almost increases in a dose dependent manner...

    So my question is:

    1> Why am I getting reference flow cell binding when I am injecting only buffer (which by the way is the commercial ready to use 1xHBS-EP)?  

    2> What I see after the injection of the different doses of the drug is it actually the drug binding to reference  flow cell or the remains from the buffer itself which keeps getting accumulated after every inject?

    hope this is a bit more clear 

  • Juvid Aryaman added an answer:
    Mitochondrial membrane potential against mtDNA content

    I'm trying to identify factors which contribute to heterogeneity in membrane potential, and was wondering whether mtDNA content could be one. I was wondering whether there exist measurements of mitochondrial membrane potential, at different levels of mtDNA content? MtDNA content can be either copy number, or different levels of heteroplasmy for a given mutation.

    Juvid Aryaman · Imperial College London

    Followers of this thread may be interested to read

    http://www.ncbi.nlm.nih.gov/pubmed/22586215

    which shows that mtDNA copy number is one of the poorest correlates of mitochondrial content in a physiological setting. So at least for perturbations on the scale of endogenous fluctuations, one might not expect to observe any change in membrane potential at all. However, I would still be interested to learn more about the consequences of copy number perturbations outside of the physiological range, or the effects of heteroplasmy.

  • Fatma Alrashidi added an answer:
    Does anyone have experience in growing mouse Beta-TC-6 cells?

    I need to grow mouse Beta-TC-6 in the lab, I searched for the medium protocol but did not find any published data?

    Do anyone have an experience culturing them?

    what is the protocol of the medium?

    should I add 2-Mercaptoethanol to the growth medium?

    Thanks

    Fatma Alrashidi · University of Bristol

    Dear Matteo

    Thank you for your replay ... it was of help

    Fatma

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