Cell Biology

Cell Biology

  • Ami Tamir added an answer:
    How can I separate live and dead Jurkat Cells?
    I recently thawed jurkat cells and counted with trypan blue. However I saw much more dead cells. I would like to separete the live ones. How can I do this?
    Ami Tamir · Insight
    As Chris Weston said, but you can use Ficol Good Luck
  • Valeria De Arcangelis added an answer:
    Can any one please tell me that specifically which constituent in FBS inhibits trypsin-EDTA activity?
    I work with adherent mammalian cells and during passage have to add trypsin -EDTA to form the cell suspension. After this 10% FBS containing media is added to neutralise the Trypsin -EDTA activity. However the constituent that is responsible for inhibiting Trypsin activity is not known to me....can any body please help asap?
    Valeria De Arcangelis · Catholic University, Rome Italy
    Interesting question Vincent. My mentor told me to inhibit with DMEM-FBS, 1:10 respect the trypsin, without any further explanation, without adding tripsyn inhibitor (here an example http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/trypsin/trypsin-inhibitors.html). I think that the inhibitors is needed, it is not only a saturation effect, but it is only my opinion, maybe we need help from a biochemist!
  • Laurel Bate-Eya added an answer:
    How should I go about plating SH SY5Y cells in a 96 well plate?
    Hi all, I am hoping someone has tips on how I should plate my SH SY5Y cells into a 96 well plate as I have limited experience with these smaller wells. Is 5000 cells an adequate amount to plate? And if I leave it for say 24hrs, would the cells not become overgrown by then? Any current protocols that work would be much appreciated.
    Laurel Bate-Eya · Academisch Medisch Centrum Universiteit van Amsterdam
    SY5Y cells 5000 cells in 96 well plate works out fine. We work with a lot of neuroblastoma cell lines and seeding densities of these cells in 96 well plates have been optimized. The it is also important to note that seeding densities of your cells will depend on how long you want to perform the experimen. Hope this helps
  • Bassam Hamdar added an answer:
    Should basic science research compromise significantly to win more fund support?
    Research in applied sciences is becoming more and more popular in attracting funds especially in biological fields. Since no research lab can survive without grant support, they are gradually drifting towards applied biotechnology, nanotechnology, drug development studies and preclinical/clinical trials. Should science solely be dictated by the demand of the market or scientists should have greater responsibilities for significant basic contributions that could help progressing individual field towards a new horizon that could ultimately change the face of the subject, redefines explored areas and opens prospective new avenues.
    Bassam Hamdar · American University of Science and Technology Lebanon
    Greetings, Research should be based on a need to discover new theories or to develop existing ones. The developed world with the huge financial dedication to research is more inclined to conduct a basic science research. However, the developing world with meager funds to research and with corruption levels sky rocketing is more able to conduct applied research. Best regards.
  • Ercument Dirice added an answer:
    Does anyone have a protocol for freezing human Langerhans Islets?
    I need to freeze isolated islets from human pancreas. The islets grown in CMRL 1066 medium supplemented with added vitamin E, nicotinamide, heparin, albumin, and ciprofloxacin. Some authors use RPMI 1640 with FCS and 10% DMSO, others use Medium 199 with added FCS and 10% DMSO. With my islets growing in CMRL 1066, I would like to know if I can use the same medium for freezing just by adding FCS and DMSO.
    Ercument Dirice · Joslin Diabetes Center
    After we receive human islets we culture them O/N in Miami Media. Next day we handpick all healthy islets and use them in different experimental protocols. If we need them for expression analysis, we handpick islets with its media (on ice), wash in PBS (x2) and quickly snap-freeze in liquid nitrogen.
  • Andrey Golubov added an answer:
    How can we express and isolate talen protein?
    We custom-designed talen activator sequence and it is in a vector. I need to express this protein and need to isolate it. I do not know the nature of protein. We have pet 45b expression vector t7 inducing competent cell for expression. I need suggestions regarding the problem and caution along with expert advice and if possible some protocols for expression of talen (even a good pdf may be useful).
    Andrey Golubov · University of Lethbridge
    Hi Rakesh, How did you clone TALENs into pET45b? Regards, Andrey
  • Mark K Chee added an answer:
    Advantages and disadvantages of sodium azide/thymol/thimerosal?
    Typically most labs use sodium azide as a preservative, but there is also thymol and thimerosal that are also well-known preservatives. Is there a reason, why azide is used much more often than the others? Does anybody know a rule of thumb for when to use which preservative?
    Mark Chee · Duke University
    I have also come across 20% ethanol as a preservative for affinity purification resins, including protein A-Sepharose, which I prefer to use when possible.
  • Alessandro Vannozzi added an answer:
    Is there any chemical which can be used as jasmonate signaling pathway inhibitor?
    I'm trying to look at the induction of candidate genes involved in plant defence when I block the signalling pathways related to JAs and ethylene and apply different stresses. For ethylene I can use the AVG, an inhibitor of ethylene biosynthesis, but as far as I know, there are no inhibitors of the jasmonate signaling pathway. Do you have any info about it?
    Alessandro Vannozzi · University of Padova
    Hi Gustavo! Thank you very much for your help!
  • Roman A. Romanov added an answer:
    Purinergic receptor stimulation?
    I've to perform patch clamp exps on CaCC after stimulation of purinergic receptors. I've seen both APT or UTP is used, which one is preferred? I know that ATP solutions are quite instable and ATP rapidly hydrolizes to ADP and Pi, does UTP have the same problem? Or how do you solve the problem of purine solution stability? I cannot use them ice cold in the cell. Any suggestions?
    Roman Romanov · Karolinska Institutet
    Dear Maria, It is difficult to add a new comment after the comment from Prof. Burnstock:) As for our experience - we prepared ATP stock (10mM in water), aliquoted, froze and use those aliquots 2 weeks at least. Having the stock solution you can change "old" ATP (2-3 hours) to a fresh one in a minute. Best Roma
  • Andreas Eisenreich added an answer:
    How to determine which transcript variant of a particular gene is differentially regulated after performing a microarray analysis?
    I want to clone the transcript variant that has the greatest fold change. Is there a way to get this information from the Affymetrix gene expression analysis files?
    Andreas Eisenreich · Charité Universitätsmedizin Berlin
    By using exon arrays or by NGS
  • Pawel Buczkowicz added an answer:
    Can anyone suggest me the best article regarding histopathological grading of brain tumors specially glioblastoma?
    I had few but not in details.
    Pawel Buczkowicz · SickKids
    I agree that "The WHO classification of Tumours - Tumours of the Central Nervous System" is your best bet. However for general overview check out: http://www.ncbi.nlm.nih.gov/pubmed/8586458
  • Kwon Paul kwang ho added an answer:
    If I treat cycloheximide to polysome fraction, then ribosome will be dissociated or not?
    I am not sure that cycloheximide has an effect on ribosome dissociation.
    Kwon Paul kwang ho · Pohang University of Science and Technology
    Thank you for all the great answers. ^^
  • Sara Parker added an answer:
    Mammospheres washing off matrigel
    I am trying to fix and stain mammospheres formed on matrigel in chamber slides using MCF10A cells. I am having difficulty retaining the formed mammospheres on the matrigel. All steps have been performed at room temp, using room temp reagents. I have been removing the media (and all subsequent washes/incubations) using a pipette (not an aspirator), fixing cells in 3% PFA for 30 min (3X PBS wash), permeablising in 0.5% T-X100 for 10 min (3X PBS wash) and blocking in 8% BSA-PBS + 0.01% Tween20 for 30 min (3X PBS wash). This is followed by RT Ab incubation, primary 3 hours RT, secondary 1 hour RT, before removing plastic wells and covering slide with a glass coverlip. However, from looking at the cell pre fix/stain to what I see on the scope, there are far fewer (prob 10% of original pre fixed) mammospheres remaining. Any clues as to what is causing it, what may help? I havent looked too closely at the number of cells remaining after each step so can't tell if its a cumulative thing or one specific step wipes them out. Next time I fix and stain some I will pay more attention.
    Sara Parker · The University of Arizona
    Thank you so much for asking this question, Chris, and for answering everyone! I just did a side by side comparison of 4% PFA versus 4% PFA + 1% gluteraldehyde, and the difference is amazing: globs of matrigel and MDCK cysts are floating about in the PFA fix but everything stayed put with gluteraldehyde. So weird! I hope the background fluorescence isn't too awful, but will try the sodium borohydrate
  • Sachindri Rana added an answer:
    Does freeze drying change enzymatic or non enzymatic activity of antioxidants in plant tissues?
    I want to freeze drying plant samples and after that measure the activity of antioxidants in plant tissue. Is it maybe?
    Sachindri Rana · Vels University
    Hi Vahab, I did not freeze dry my samples. I just stored them at -80 degrees Celsius and used them for the analysis. I stored them for almost for a month and then used them all.
  • Manish Joshi asked a question:
    Are polyubiquitinylated proteins targeted to proteasome from both 19S Caps?
    As we know that proteasome is a 26S complex. It contains a 20S core barrel shape structure and a 19S cap on both sides of this core, and polyubiquitinylated proteins are targeted in the proteasome through the 19S Cap. Are the polyubiquitinylated proteins targeted from both of the 19S Caps, or there is any specific mechanism for a one side only?
  • Siva Kumar added an answer:
    What is the difference between Fasta sequence and Atton Seatedquence?
    Can anyone explain the Fasta and Attonated gene sequences? Are they the same sequence or different?
    Siva Kumar · Sri Sankara Arts and Science College, University of Madras
    First i want to correct the question, i hope it is the difference between fasta and Annotated sequence instead of fasta and attonated sequence. Both fasta and annotated sequences are different formats of same sequence. You can refer it from the following links: Fasta format: http://www.ncbi.nlm.nih.gov/nuccore/327323284?report=fasta Genbank format: http://www.ncbi.nlm.nih.gov/nuccore/HQ874461.1 The main difference between two formats are that fasta is simple format whereas annotated sequence formats have more details or information about sequences. Genbank is one of such annotated sequence formats.
  • Neil A Bradbury added an answer:
    Does anyone know why my 16hbe cell lines die during starvation?
    I work with particulate matter and prior to treatment I serum starve 16hbe's (bronchial epithelial cell line) to synchronize them. Prior to starvation the cells are allowed to grow to 70% confluence. Following starvation, cells lift off the plates and are observed suspended in the media. I have changed the well sizes (48 to 12 well nucleon cell culture plates) and the media (1% BSA in mem glutamax to ultraculture) yet the same problem persists. Can anyone tell me how to get around this?
    Neil Bradbury · Rosalind Franklin University of Medicine and Science
    16HBE are not the best of cells to stick down anyway. One thing you could try is using conditioned media from 293 cells grow in serum free media. filter it and use that as your growth media. Also make sure u are using coated plates to help the cells stick.
  • John R Henley added an answer:
    Does netrin-1 act as a chemotactic guidance cue for myeloid cells?
    Does it act in a similar fashion to the function of netrin in axon guidance? For example, does it have a role in attracting axons of spinal commissural interneurons to the ventral midline, or does netrin have a distinct function on myeloid cells? Is there evidence of microglia homing to sites of netrin production in the developing nervous system, perhaps at the ventral floor plate? This is a fascinating paper and I would be grateful for additional insights.
    John Henley · Mayo Foundation for Medical Education and Research
    I tried asking this question previously but appear to have been unsuccessful in getting it to post. Apologies in advance if this is a duplicate. I am enthusiastic about learning more on this topic.
  • Greeshma Venugopal added an answer:
    Can anyone help with isolated feline PBMC?
    I have been isolating PBMC's from feline blood with lymphoprep and it was all working fine. Now I still get good layers but when I centrifuge the isolated cells they clump and smear on the bottom of the tubes. We have just started using the new corning centristar tubes and have a new batch of FCS, both of these I will evaluate. Any other thoughts ?
    Greeshma Venugopal · Sandor Life Sciences, Hyderabad
    sorry for this late reply. but please check with lymphoprep too. though it is clumping you can remove the smear and work with the remaining cells. If you carry further with some assay, keep a calculation in mind about the cells loss and take extra cells accordingly. and also check blood collection quality and also breed of cats etc.
  • Bernie Cohen added an answer:
    Can we define organisation and formalize it in a manner that supports the explanation of life phenomena?
    Organisation, its omni-presence, preservation and dependence, is possible the distinctive aspect of biological phenomena. Achieving a consensus about what is organisation and how can it be used to generate biological explanations would greatly benefit the development of (theoretical) biology.
    Bernie Cohen · City University London
    The enterprise modelling framework called 'Projective Analysis' (or PAN) provides a structure for organisation that is consistent with the reflexivity in Rosen's categorical model, with the third order cybernetics in Maturana's later work and with the Lacanian/Freudian accounts of consciousness. It also provides a full set of computational tools for eliciting, composing and analysing people's models of the organisations they inhabit and for indicating the 'holes' in these models. See the papers and blogs at www.brl.com and www.asymmetricdesign.com.
  • François Bouteau asked a question:
    In FMRF amide related peptides, amidation is allowed by the terminal glycine. What could be the role of a tryptophane in place of such glycine?
    As above.
  • Ridha Limame added an answer:
    Is Epithelial-mesenchymal transition (EMT) a dedifferentiated state of the cell?
    EMT generate cells with stem like properties. I was wondering whether it reverses the the differentiated cells into undifferentiated state? My cells undergo dedifferentiation and acquires cancer stem cell like properties but it doesn`t induces EMT and it loses clonogenicity.
    Ridha Limame · University of Antwerp
    I think it must be noted that EMT is a transdifferentiation program, i.e. cells indeed dedifferentiate from an epithelial phenotype (loss of adherens and tight junctions, cytokeratins, cytoplasmic ß-catenin), but other genes show a concomitant increase of expression (vimentin, fibronectin, N-cadherin) leading to a mesenchymal differentiation. The story is not complete by just explaining EMT as a dedifferentiation process.
  • Jijo C Joseph added an answer:
    What is the best method for isolation of mitochondrial DNA from tissue samples?
    I'm trying to isolate the mitochondrial genome from tissues of marine invertebrates. Initially I tried to use a kit (ABICAM) for isolation of mitoncondrial DNA, however there is considerable contamination with nuclear DNA. Does anyone have a reasonable option for this?
    Jijo Joseph · Central Marine Fisheries Research Institute
    I think tek buffer method will help to isolate mitochondrial dna isolation
  • Laura M McLane added an answer:
    Does anyone know what this nuclear structure is?
    I created a GFP-fused mutant of my favorite transcription factor and expressed it in 293T cells. The mutation is in the putative DNA binding domain. It is not co-localizing with regions of dense chromatin. The wild type protein actually appears to be excluded from these structures. Any insight would be great!
    Laura McLane · University of Pennsylvania
    Thank you for your responses. The GFP is under the CMV promoter so it is strong, however, the wild type version of this protein (which is two amino acids different than the one imaged above) does not aggregate. It is exclusively nuclear and, in fact, it is actually excluded from these foci. I'm not sure it is aggregation but it is certainly possible. As for staining for the endogenous protein, it isn't expressed in 293T cells. I think I will try co-staining for other markers as suggested. The IP is a great suggestion. And so is the alternative tag. I will look in to doing both. I am just baffled by why a DNA-binding mutant that otherwise associates with active chromatin would be relocalized or sequestered into these foci. Thank you again for your suggestions!
  • Vello Tõugu added an answer:
    What does precisely intrinsic activity of the protein mean?
    Currently I'm reading a lot of literature concerning one protein. Everywhere I look, they are talking about intrinsic activity as an important feature for the activity of the protein. As well, everywhere I looked, I didn't quite understand the term. Could someone explain me what does it precisely mean and what happens if protein has a low or high intrinsic value. Thanks
    Vello Tõugu · Tallinn University of Technology
    If the intrinsic means that no other molecules are required, then which term we should use to compare the activity on two isoforms of an enzyme in teh presence of a cofactor?? In the case of enzymes Vmax is "per mg" and kcat is "per molecule".
  • Simonetta Palleschi added an answer:
    Why don't we use confluent cells in experiments?
    This may seem a stupid question, and might actually be one: I'm familiar with cell culture and do experiments with cells. It is widely know that most mammalian cell lines cultured in the laboratory, as most cells, follow a growth curve with lag, log and plateau (and later death) phases. It makes sense for production to split them in the log phase, when they are actively dividing and colonizing the available space. But when it comes to plating them and performing experiments, it seems that we have inherited that rule: virtually all of the experimental procedures that can be found in papers are performed with cells between 60 and 85% confluency. Asking around the laboratories, everybody seems to think that using confluent cells for experiments is something that should be avoided. Of course, the growth curve itself makes evident that confluent cells and log phase cells present very different behaviours, but I'm not able to recall what were the reasons that lead researchers to decide that the only relevant ensemble of cellular states was the log phase or that it was preferable to the others. In fact, we use cells with traits of cancer cells (when not directly cancer cells) because of practical needs (easy, sustained and humane production of cells for research) but we frequently use these systems to try and draw conclusions about general cellular biology. But most of the time cells in mammal bodies are not cells actively dividing and colonizing available space (those would be cancer cells and cells during development and wound healing) and one could think that cells between the end of the log phase and the onset of the death phase might be more reminiscent of cells in their physiological state. So why avoid using them? Or I am wrong and there is a significant body of research and knowledge on the biology of confluent cells that I haven't come across to?
    Simonetta Palleschi · Istituto Superiore di Sanità
    I agree with Patrick and Brandon. With few exceptions, studies with confluent cells reflect more physiological conditions than those with sub-confluent, still rapidly growing cells. However, since confluent cells in a dish are still very far from a "physiological normality", one should select for the exps the right time window after the confluence is reached – so that cell-to-cell interactions are maximized - and before cells start suffering and dying
  • Sarah Wright added an answer:
    Advice on high content imagers: Perkin Elmer Operetta vs Molecular Devices ImageExpress Micro?
    Does anybody have any advice on the quality and functionality of the two high content imagers and their associated analysis software above? Image quality, ease of analysis, reliability etc. Many thanks in advance!
    Sarah Wright · iPierian Inc.
    I have only used Molecular devices ImageXpress Micro and Ultra but think it is a very reliable system with good images. It is easy to view plate thumbnails and do analysis.
  • David Ian Leavesley added an answer:
    Are RGD (Arg-Gly-Asp) peptides the right choice to block integrin-ligand interactions?
    I am searching for a pan-inhibitor of the integrin signaling pathway. Would usage of the RGD (Arg-Gly-Asp) peptides be the right choice?
    David Leavesley · Queensland University of Technology
    Agneiszka. This depends on whether your objective is to inhibit ALL RGD-dependent interactions, specific integrin interactions, or specific ligand induced pathways. Not all integrins use RGD as their sole target motif, and not all RGD's motifs are functional. (e.g there is one in BSA!) Equally integrin-induced intracellular signalling cascades are not singular nor unidirectional. I think you need to think carefully about the question you are asking and then devise an appropriate strategy. And please remember that when you block what you think is a unique target, it is highly unlikely it will function in isolation. Thus there WILL be off-target effects! How do you discriminate these from your objective? Put simply, it isn't as simple as you would like to believe! David L.

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