- Rostyslav Krutyholova added an answer:6Positive control for cycloheximide and MG-132I've treated my ovarian cancer cells with two inhibitors which are cycloheximide (inhibit denovo protein synthesis) and MG-132 (Inhibit proteasome degradation). What I would like to know is what can I use as a positive control to confirm that these inhibitors worked? I'm doing Western Blot with the cell lysates.
I would suggest beta-catenin, normally this protein is degraded in proteasome, b-cat possess a short half-life so you will be able to see accumulation!Following
- María Clara Carou added an answer:13How can I prevent edge effect in TUNEL assay?
I am recently conducting TUNEL on (perfused) mouse brain cryosection and so frustrated by its edge effect in DNase-treated positive control (The attached photo is a closeup of edge area). Not as expected to be green everywhere, inner region of the slice has little signal.
I have tried to adjust duration of triton incubation, amount/concentration of DNase and also temperature of DNase incubation but no improvement so far. So could anyone give suggestion what can cause the effect and how to troubleshoot next?
Thanks so much for your help.
Be sure Tritón X100 is fresh. You have to prepare permeabilization medium just after use it.Following
- Nirmalya Dasgupta added an answer:17What 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.
The Pubmed link of my paper-
If you want I can send you the earlier figure also, where objection was raised.Following
- Alexander Abrams added an answer:5Does mitofusin promote or inhibit contacts between mitochondria and the endoplasmic reticulum?
A recent report by Filadi and colleagues reports that removal of mitofusin increases mito-ER contacts. This is interesting but puzzling. Schneeberger and colleagues (PubMed ID 24074867) also perform quantitative analysis of mito-ER contacts in vivo, in neurons, and reach the opposite conclusion: Loss of Mfn2 decreases mito-ER contacts (see Fig.6G). Any thoughts from the mito experts?
I think that the answer to that question is heavily debated. Mfn2 is important for mitochondrial morphology but it is hard to say if it actually mediates direct contact with the ER or if its apparent role is secondary to mitochondrial morpholy. ER-mito contact is most accurately measured through 3d Em, as confocal seems to be insufficient and tem is too low through put and potentially biased.Following
- Maurizio Pea added an answer:4What 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.
Kinetic data obtained at neutral pH suggests that ketimine reductase/CRYM plays a major role as a P2C/Pyr2C reductase and that AECK is not a major substrate at this pH. Thus, ketimine reductase is a key enzyme in the pipecolate pathway, which is the main lysine degradation pathway in the brain.Following
- Venil N Sumantran added an answer:22What is the best cell line for glucose uptake assay?I have been trying to do a glucose uptake experiment using fluorescent dye to measure if my protein of interest will have impact on glucose uptake. From experience, what is the best cell line for this? Do you add insulin? Do you starve your cells?
I have not doen this assay, but its best to start with an insulin dependent cell line to prove you have active glucose transport. This can be your positive control and you can standardize optimum conditions to visualize your fluorescent probe. Then, you can work on your protein of interest.Following
- Swati Kumar added an answer:9Have you ever tried to transfect THP-1 cells? How high is the transfection efficiency?I have tried to transfect THP-1 cells (human monocytic cell line) with a plasmid containing the GFP reporter gene but I could not get a high transfection efficiency. I also tried electroporation (Neon Transfection System) and the transfection efficiency was higher. Although the cell viability was lower, almost all of the cells alive were transfected. I analyzed only by fluorescence microscopy but I could analyze by FACS.
I would like to know if there is someone used to work with THP-1 cells and if I could get some tips about how to work better with them.
Hi Veronica, I have been having poor survival with neon with just about all their conditions in the optimization protocol. I was wondering if you could share what conditions you've used to transfect with neon -voltage and pulse number wise ? Any insight would be greatly appreciated.
- Vally Kommineni added an answer:5Cell Based Assays........Can some one tell me about Cel base assays ??
How it is different from invitro assays????
Plz tell me in the brief for understanding.......
Cell based assays qualitatively measure the function of the product/molecule. Assay plays a key role in determining the quality of biological products. Potency can be determined with relevant cell-based assays, not just by one assay. Assay designing depends on molecule's MOA. In simple terms determining drug quality before risking in pre-clinical studies.Following
- Tamas Varga added an answer:14What are the good markers for G2/M phase of cell cycle?I was getting high G2/M population in PI staining. I was looking for a specific marker (FC/IF/WB) of G2/M transition. Cyclin B1 and CDK1 usually acts in this stage. Can anyone please suggest a marker?
To those who raised the ploidity issue: does the endoreplication (e.g. in Vincent's reply) occurs only in cancer cells? What drives endoreplication in these cells?
- Mubing Duan added an answer:14Anti-CD11c mouse Ab working in paraffin-embedded sections?We have been trying to get by IHC labeling of CD11c positive cells in our lungs and spleen sample from mice.
The best we could get was a weird labeling in red.
We do not label the cells as the labeling is smaller than the nucleus.
It seems that our Ab is being trapped in some particular unknown structures...
So right now, I am not trusting this can of picture to say that I'm seeing CD11c cells.
Here the following info to understand the pics:
it is confocal microscopy: depth of the plan: 0,125 um.
blue is DAPI
green is an FITC-labelled Ab
The 2 pics show you different fields.
Thanks in advance for your help.
The most traditional PFA fixed and non-paraffin embedded method would be be cryosectioning (but frozen sections are very difficult to cut, and there is a loss of architecture - the upside is that better antigen preservation exists so sometimes people are forced to go down this route). We are also actually exploring other ways in the lab of staining PFA-fixed lung tissue (cutting 150 um thick agarose gel embedded sections on a vibratome for example).
It sounds like your samples are all paraffin embedded samples though. Unfortunately, I have come across very few papers with CD11c staining in paraffin sections (I have seen them for MHC Class II though in mice, could this be an alternative for you?). I have seen CD11c staining for cryosections though (http://emboj.embopress.org/content/31/10/2378), which makes me suspect it is an antigen which gets masked or destroyed by the embedding process.
I've attached some old staining of mine on CD11c staining of PFA fixed alveolar macrophages ex vivo undergoing phagocytosis. The blue is a typical pattern of CD11c staining seen (on the surface rather than internally - in this experiment I tried to fit 5 markers into a 4 colour confocal microscope, so my DAPI/BV421 channel had both Hoerchst internal and CD11c external staining).Following
- Waleed Renno added an answer:7How reliable is NeuN for labeling neurons? Is it possible it labels other cells too?I am not sure if the possibility was ever exhausted that it labels other cells like glia.
A combination of at least 2 to 3 different staining of neuronal markers need to be always used to confirm the immunohistochemical analysis of neurons in addition to Cresyl violet (Nissel Body) staining. One can use any combination from the list below:
b-tubulin III , microtubule-associated protein-2 (MAP-2), neuronal nuclei (NeuN),
neurofilaments (NF) , NF [PAN, clone: DA2/FNP7/RmdO20.11 (PAN)] , and neuron-specific enolase (NSE).Following
- Usha Yadav added an answer:3Does anybody know of a paper regarding how stable human cells are during M phase as compared with G/S phases?
I was taught in school that cells in M phase are "weaker" and more prone to cellular collapse if stressed. I find dividing circulating tumor cells in patients, and am looking for publications on whether this should be expected, or if the structure should have collapsed do to the stress in circulation?
You are right M-phase cells are more prone to damages because of doubled DNA content, if DNA Damage in normal cells crosses certain limit cell prefers to die and start programmed cell death process on its own and such dying cells are cleared by immune cells from the system. In this regard cancer cells are also a very sensitive to DNA damage(as they divide very fast) and their repair process is also not very accurate. But, at the same time lot of cancer cells if not all develop mechanism to avoid programmed cell death process even having lot of damaged DNA content and they also escape our immune system. That's why cancer cells always have higher level of DNA damge, chromosomal aneuploidy or polyploidy. Many of them die too but again division rate uncontrolled so it persist and grow....Following
- Rida A.Q added an answer:5How can we store JC1 stained cells for FACS analysis after a week ?
Actually, I stained cells with JC1 dye, but I found that FACS was not working.
Could anyone please tell me that how can I store these samples for future FACS analysis?
Thank you so much for the answer Elizabeth Sloan and Heirberto.
Yousef Abdulrazzaq: I don't fix the cells and thank you for the answer.Following
- Aleksandra Somogyi added an answer:10Why 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?
Hi Andrés, there is also a cerebellar granule precursor cell model of the neurodegenerative disease JNCL where cells are grown in confluency for 7 days so they build the lysosomal storage material that is typically found in patients (Fossale et al 2004). In my opinion, this treatment mimics better the stress/condition they have in real tissue whereas still proliferating cells allow us to seperate early phenotypes from symptoms of the mutation.Following
- Wu Anguo added an answer:49What is the best percentage of gel for a 14 kDa protein using western blot and the best voltage for transferring the protein from gel to membrane?Running a western blot gel using a 14 kDa protein.
12% gel, I usually use 12% gel to analyse 14-16 kDa protein such as LC3Following
- Pinar Kanlikilicer added an answer:3What is the best way (or software?) to count cells after fluorescent and DAB IHC staining?.
I also use IHC Toolbox plugin to get the image with just DAB staining by simply selecting "H-DAB model" and then "color" on that plugin . Now, you have an image consisting of only brown stainings , nothing else. Then you can do the followings:
Image>Adjust>Threshold ( Now, all brown stainings are in red)
You should play with the parameters. For CD68, I used 30-infinity, 0-1, outlines. It worked well for my images. Finally, you have all the counting.
Hope this helps.Following
- Lizbet Todorova added an answer:9How long does it take for cells to secrete extracellular matrix proteins?
Hello everyone, I am designing an experiment to investigate cell secretion of MMPs. My main question is, after how long can one detect secreted protein? I have also observed that researcher either use medium or protein sample to carry out western blotting for MMPs. I would like to know which of these methods has the best results and why.
You can also perform a proliferation assay with the exact cell amount you are going to use for MMPs assay. If you are using the supernatant to detect the secreted MMPs then you can relate to protein amount in the cell layer.Following
- Katie Owens added an answer:11How to prepare Phenol red stock solution?
I need a 3mg/ml stock solution.I tried to dissove with ethanol/methanol; but in 2mg/ml also it's not soluble?
I need to make acetic acid standard curve using Phenol red indicator.
I know this is an older feed but I ran into this problem today. Using phenol red, I figured out the formula to use NaOH. Then realized we actually buy the phenol red sodium salt itself.......Following
- Alfredo Cabrera-Orefice added an answer:3Can anyone tell me the difference between the isolation of enzymes from mitcochondria and peroxysomes?
Can anyone tell me the difference between the isolation of enzymes from mitcochondria and peroxysomes?
Even in isolation
Your welcome... best regardsFollowing
- Gal Haimovich added an answer:8How to selectively kill MEFs fast?In a mixed culture of immortalized MEFs (Large Tag), I need a way to kill a *specific* subset of the cells fast (hopefully within 1-2hr). So, I am looking for a drug or treatment that can kill MEFs fast but also to transfect MEFs with some kind of resistance gene so I would get a selective killing of only the untransfected cells in the mixture (or vice versa- transfect the cells I want to kill with a pre-conditioning gene that would make them highly sensitive to the treatment). I tried G418 but after 3 hrs, there is little death even at a high concentration. That is too slow for me.
If anyone still follows...
I tried KillerRed. In my hands, my cells (immortalized MEFs) two types of promoters (CMV or Ubc) and several microscopes for variable exposure times (2->20 minutes) - didn't work (cells just didn't die).
I am now trying diphtheria toxin to selectivly kiil human cells in a mouse-human co-culture. so far, some cell lines are resistant (U2OS) and the others just die too slowly for my purposes (H1299 ~50% after 30hours, HEK293, HEK293T ~10-20% still alive after 30hour).. But, am still trying..Following
- Srinivas Thota added an answer:11What is the best method for ribosomal RNA depletion for Drosophila RNA seq?
We would like to make RNA-seq libraries from Drosophila RNA. It is important that we deplete rRNA rather than enrich for Poly(A) as we would like to analyse some non-polyadenylated species in our datasets.
I have read that the Illumina Ribo-Zero rRNA depletion kit (Human, Mouse, Rat) is reasonably effective, although does not fully remove 5S or 28S (right arm) rRNA.
I'm also thinking about an rRNA "RT blocking" rather than "depletion" strategy a la the method presented in the attached paper, although I would have to devise a strategy for removing all types of rRNA (not just 2S).
Has anyone had experience with this, and would you please share some wisdom? What sort of parameters would I have to be careful to consider using these approaches? Do you have alternative suggestions?
As an aside, these RNA-seq libraries are likely to be from low-input RNA, and the quality could be less-than-perfect as we are isolating RNA after formaldehyde fixation.
Thank you in advance for your intelligent answers.
Can you please update your progress?Following
- Lei Hong added an answer:14Can anyone help with endosome isolation?I tried to isolate early endosome from normal Hek 293 cells by using two step ultracentrifugation.
For this I first homogenised the cells and collected post nuclear supernatant and store this PNS at -80.
After two days I prepared sucrose solution.
Firstly I loaded the centrifuge tube with PNS adjusted to 40% with 62% sucrose solution. Then overlaid with 1.5 ml of 35%, then 1ml of 25% and finally fill the tube with HB buffer.
Ultracentrifuged at 35000rpm in swinging bucket rotor for 1,5hr.
But after centrifugation I could not find any interphase.
I am wondering what mistake I have made.
In addition to this I have two other queries also:
1) Can we store PNS? If yes then at which temp?
2) Is it is essential to measure the refractive index of each sucrose concentration before making gradient.
I saw an interphase at 35% sucrose. however, the 25% sucroser interphase can not be detected.Following
- Im Aparicio added an answer:18Why does DAPI staining not detect mitochondria?
We suspect mycoplasma contamination in one of our cultures since DAPI staining reveals cytoplasmic staining. But one person suggested it was simply mitochondrial. This got me thinking: in all stainings we have done and in all staining I have seen published where the investigators performed DAPI staining to reveal the nucleus, why do we never see mitochondrial staining? It should reveal many cytoplasmic structures yet we don't see any.
I'm working with spermatozoa. Mitochondria, in these cells, are located exclusively in the midpiece of the tail. When I use DAPI, only the head of the sperm is stained.I have never seen the mitochondria of the midpiece stained with DAPI. Hope it helpsFollowing
- Xinyan Tang added an answer:11Is it possible to differentiate between cells that were alive/dead prior to cryosectioning?
I am trying to section tumor spheroids as I need to be able to see what is happening on the inside of the spheroid. For my research need to be able to distinguish which cells were alive and which were dead prior to the sectioning. Right now I am staining the spheroids with DAPI and trypan blue as a live/dead assay (limited budget, need to use what is available at hand) but can't seem to stain more than the first few layers of cells.
I used Live/dead assay in Invitrogen/life technology for staining cells in a 3D alginate spheroids before cryosectioning. It was very good to look at the live/dead at all "layers",But not sure how much it can penetrate your tumor spheroids sample. you may want to try. It is very easy and straight. Good luck!Following
- Siamak Gholamalipour added an answer:1what kind of adenosine receptor (A2A , A1) is activated in responding to the secretion of ATP by astrocyte?ATP released from astrocytes is degraded to adenosine and activates presynaptic adenosine A2 or A1 receptors that leads to an increase or decrease in its release probability (Panatier et al. , 2011). Now the problem is:
After secretion of ATP by astrocyte:
Which mechanism is activated A2A receptor on presynaptic neuron?
Which mechanism is activated A1 receptor on presynaptic neuron?
Which mechanism determines that what kind of adenosine receptors on the presynaptic neuron (A2A , A1) should be activated in response to astrocyte adenosine secretion?
please check this article
I hope it's helpful .Following
- Siamak Gholamalipour added an answer:3How big is a gut epithelial cell (enterocyte)?Does anyone have a good handle on what capacitance epithelial cells are? Are neuroendocrine cells bigger or smaller?
You are welcomeFollowing
- Gozde Akdeniz Skvortsov added an answer:12How can I prevent all the cells from emitting fluorescence in the red channel in a Live/Dead Assay of Microtissue?I'm performing the viability Live/Dead assay on Microtissue (high cell density of cells - 250,000 cells/ mm3) using Calcein Am and Propidium Iodide and imaging them using a confocal microscope (488nm Exaltation Laser). I intend to finally count the number of live and dead cells using Imagej.
Now, thought the experiment works perfectly well for cells on a glass slide, they won't work too well with the microtissue (Using a z- stack with 2um steps). The green channel cells show (99%) cells to be green. The red channel (Propidium Iodide) shows (100%) cells to be dead. Though you should just be able to 1% of the dead cells in the red channel. (Picture attached)
How can I prevent all the cells from emitting fluorescence in the red channel? Am I using a high concentration of dyes? Or is it just hard to image cells in 3D culture?
Is there any other assay for cell viability?
Hi Naveen, thanks. I do not have any other option apart from the confocal so I have to give it a try with the confocal. Hope I will also find a way like you did :)Following
- Pardha Saradhi Attuluri added an answer:8Which red-fluorescent protein can work in the extracellular space?
We are trying to generate a secreted red (or far-red) fluorescent protein construct in Drosophila. We had made some years ago such a construct with mRFP fused to a signal peptide, which worked OK, i.e. red fluorescence was detectable in the extracellular space, although signal was quite sensitive to bleaching. Now we tried to make a brighter and possibly more photostable version by fusing mKate2 as a fluor to the same signal peptide sequence that we had used successfully before. This construct does not appear to give any detectable fluorescence. We're not exactly sure what the problem is, but were suspecting that mKate2 might not fold or mature properly in the secretory apparatus, or that an oxidizing environment might affect the fluorophor. Does anybody have an opinion or experience on this? Any suggestion on which other red or far-rad fluorescent protein might be a better choice in this case? Thanks!
mRFP1 and mCherry are being used to label endosoms, vacuoles and plasma membrane in plant model Arabidopsis thaliana. to my knowledge secretory system's pH and oxidations states are similar in both plants and animals.
regarding TagRFP-T: this is several folds photostable compared to its predecessor FP TagRFP but lil less brighter. If you think your protein is very abundant in extracellular space go ahead with TagRFP-T otherwise TagRFP would be better choice.
the reason for low fluorescence of your mRFP construct can be due to low number of molecules reaching extracellular space (use brighter FPs like TagRFP or 2x /3x version of mRFP1) or stability issues of FP while moving through secretory path (low pH and oxidising environments) for later issue following article may give some direction to choose proper FP
all the best :)Following
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