- 2Which stem cells are best to do cell culture studies on wound healing?
I need to know whether co-culture is the best way or only one cell culture on the prepared scaffolds is a way to do for wound healing?
Both are a good way to study , And you can choose the best way through looking on the similar researches for idea of your Search and the possibility of application this method in your laboratory .
look at links & attachment here .
Good luck .Following
- Rossella Parrotta added an answer:2Does anybody have experience in deubiquitination?
I'm setting up an initial experiment to check deubiquitination in vivo. The idea is to transfect HEK293 with my DUBs of interest and then perform acidic extraction of histones, run on SDS gel and blot with anti-Ub-H2A. Does it sound correct? Are 48h of transient transfection enough to have DUB activity and see a difference in ubiquitination of H2A? Thank you in advance!
Hello Zhonghua Zhang, thanks for your answer! Yes, I was also thinking to include a treatment with MG132 so I'll follow your suggestion.Following
- Amita Ajit added an answer:2What do you think is the best economical cocktail/niche for differentiating MSCs to Endothelial Cells?
I am working on therapeutic angiogenesis and lately i have been differentiating MSCs to ECs. I have come across numerous strategies and i would like to know your valuable opinion on the most economical & effective strategy based on personal observations.
Thank you Mandolin. This is the most commonly recommended protocol. Do u think reduced serum (2% instead of 10% FCS) has a significant difference? Have you tried it personally?Following
- Rosy-Paola Cárdenas-Sandoval added an answer:24Why should we add FBS to the serum free media?We all know, during few cell culture experiments we use serum free media, we still add FBS (Fetal Bovine Serum) in some cases. We are also aware that FBS provides the growth supplements for the normal growth of cells. Are there any other reasons?
Who knows if FBS increase the collagen type I production in fibroblast monolayer culture?Following
- Zeynep Hein added an answer:19LysoTracker® Red DND-99 for time-lapse imaging in mammalian cells?I am trying to optimize LysoTracker® Red DND-99 for time-lapse imaging in mammalian cells.
50nM; 75nM; 100nM; 500nM and 1 µM
Recommended concentration: 50-100nM
1 hour incubation at 37C.
The time interval I use is 2 seconds for a total duration of 2 minutes with 4 Z stacks. Exposure time 300ms.
Most of the concentrations result in photo bleaching i.e. a constant decrease in intensity over time except for the highest concentration of 1 µM.
Would using such high concentration of 1 µM result in any undesired effect on the cells? Or any kind of stress?
I have observed that LT Red photoconverts and starts emitting in green in live cells when it was first illuminated either with white light or green light (not laser light but filtered mercury lamp light). It therefore looks like photobleaching but actually shines bright green. In fixed cells, photoconversion is less. Here's also an article I've found, reporting about this phenomenon: http://www.nature.com/cr/journal/v17/n11/full/cr200780a.htmlFollowing
- Debojyoti Chakraborty added an answer:7Any advice on the transfection reagent for mouse ES cells?
Hey guys, I have try several transfection reagent (Xfect, lipofectamine 3000, PolyJet) for transfecting mouse ES cells. But the transfection efficiency is quite low. The best I can get is 50% (using polyJet) which was transfecting a plasmid with ~10kb. But I could just get this percentage once and got much lower efficiency in the other trials (using the same plasmid). I am not sure if the reagent in our hands function properly or not because some of them are quite old and the our freezer was broken down previously which may make the reagent degrade.
Could anyone share some experience in transfecting mouse ES cells? For example, which reagent you used? How big is your plasmid? And what is the transfection efficiency you normal get? Thank you!
I routinely use Lipofectamine 2000 for my transfections in mES cells and the largest plasmid that I have used is ~9kb with ~70% efficiency. This was reproducible. In my hands, 2 rounds of transfection also produced similar efficiencies without causing too much cytotoxicity. The typical numbers were, 40,000 cells, 1ul lipofectamine 2000, 100 ul Optimem (Thermo Fisher) and 1.5-2ug plasmid (preferably maxiprep) in a 12 well dish. For double transfections, forward (seeding 24h before transfection) method was used. Good luck!Following
- Sahil Sharma added an answer:6Inducing iPSC ( pluripotency ) using chemical cocktail?
Many researchers are working on inducing pluripotency for fibroblast cells using different chemical cocktails mainly epigenetic modifier and lineage inducers. i am wondering that whether all these chemicals can be added at once in the media (kept in 4 degree )and then media can be change every 3 days or does one has to change the media every time and freshly prepare the chemical cocktail each time seperately?
Thanks all for the wonderful adviceFollowing
- 3How can we detect CML Stem Cells and how can we isolate them from CML patients?
How can we detect CML Stem Cells, and how can we isolate from CML patients?
I agree with researchers , look at papers in attachment and links , will help you to detect CML Stem Cells marker & how isolate them .Following
- Jose A Morales-Garcia added an answer:5Does anyone know a good method for SVZ/SGZ-derived stem cells isolation in rat?
I am trying to isolate stem cells form the subventricular zone (SVZ) or subgranular zone (SGZ) in rat with very bad results. Thanks a lot for your help.
Great video Tianda. Thank you very much for your kind help. ¡Gracias!Following
- Cristhian Fimbres added an answer:11Which marker can I use in conjunction with Nestin for staining Neural Stem/Progenitor Cells (NSPCs)?I need to double stain for NSPCs as a confirmatory test that the cells that I am culturing are NSPCs. Please suggest a another protein that I could stain for. I have read about Sox-1/2. Would these be appropriate?
I use SOX2, NESTIN, OCT4 to stain IPS derived neural stem cells.
All cells are NESTIN+ and OCT4- but not all cells are SOX2+.
I find that cells that are "compact in colonies" are SOX2+, while the larger flatter looking cells around the colonies are SOX2-Following
- Elodie Vandenhaute added an answer:4How do I prevent cells growing through pores in transwell inserts forming monolayers on both sides of the membrane?
We are culturing epithelial cells on transwell filters (24 well format) with pore sizes between 3 and 5 µm in standard and inverted conditions. With some cell types we face the problem that cells grow through the pores of the membranes and form a monolayer also on the side of the membrane opposite to the side were the cells were seeded.
How can we prevent this?
We already tried different coatings and this does not seem to have an effect...
I faced the same problem and one solution is to cultivate your cells with medium in the upper compartment only. I know it perfectly works with 3-µm-pore inserts. But be careful: if you have your insert wet on the lower side - even just once during the cultivation time - then the cells will begin to invade the lower surface.
Just put medium in both compartments before your experiment, and then you can start with your transmigration experiment, being sure that you have only one layer of cells !Following
- 10Neurons/oligodendrocytes differentiated from mouse neural stem cells dying in culture?
Many of the neurons and oligodendrocytes that I've differentiated from mouse neural stem cells have begun to show a lattice-like formation around their cell bodies, and there are many dead cells-- does anyone happen to know what these lattice-like or bubble-like formations are?
As some background, I've plated 25,000 cells/cm2 on PLO/laminin-coated coverslips, and have maintained them in culture with appropriate supplements. They look great until Day 4 of differentiation, and then they begin to look like the picture attached. Any ideas?
Look at papers here Hope help you
- 3Has anyone used Plerixafor (AMD3100) in vitro?
I want to try using Plerixafor (AMD3100), an antagonist for CXCL12-CXCR4 binding in vitro. Could someone help me with the solvent that has to be used to dissolve and the shelf life post dilution?
Yes you can use DMSO and follow the same steps proposed by Daniel .
- Bhakti Niskama Shanta added an answer:1Can someone please suggest the cell line which expresses endogenous myocardin protein?...
Our article published in peer-reviewed Journal "Communicative & Integrative Biology". A few major points discussed in the paper:
(1) Brain is not the source of consciousness.
(2) Consciousness is ubiquitous in all living organisms, starting from bacteria to human beings.
(3) The individual cells in the multicellular organisms are also individually cognitive entities.
(4) Proposals like “artificial life”, “artificial intelligence”, “sentient machines” and so on are only fairytales because no designer can produce an artifact with the properties like internal teleology (Naturzweck) and formative force (bildende Kraft).
(5) The material origin of life and objective evolution are only misconceptions that biologists must overcome.
- Chitra Devi added an answer:14How to increase concentration of RNA and why do the ration of A260/A230 is low and how to overcome them?
Dear fellow researchers,
I am working on rat brain tissues. I need to extract RNA from here. The recommended RNA concentration is >150ng/ul (reading from Qubit). I am using Qiagen products and strictly adhere to their methods.
Thank you for all the replies.
I have solved the problem. I just stick to the method described by Qiagen protocol. I realized the my sample source was the reason for the problem I faced to. My sample sources were very limited; when I pooled my sample I managed to get higher yield and good ration of A260/A280. However, the ration for A260A230 were still not up to the range mentioned i.e.1,7 to 1.8!Following
- Carlos Ocaña Salceda added an answer:11Does anyone have experience with ELISA for LPS induced inflammation in stem cells?
We have induced inflammtion using bacterial LPS . Furthermore we ran ELISA for inflammatory cytokines using supernatant from treated cells. However, we could not get the results as expected and in line with genes expressed.
If anyone has experience with the same. Kindly share.
Yes I did. and the kinetics were not similar to those of the direct expression assayed through ELISA. But, commonly, 3h expression levels were high but not so consistent among themselves compared to 6h (somehow lower but with less variance).
The important point is that oscilations generate a time lag which may "hide" or "camouflage" the optimal time point to detect your cytokine/s. I was specifically looking for early inflammation, which is not your case, but you may need to stablish, at least superficially, the kinetics of the ILs you are studying to determine the most appropriate time for your experiments.
Figure 3 in this review shows the first 24 h kinetics of three different cytokines in cultured tubular epithelial cells against two different stimuli (TWEAK and TNF-alpha), just so you get an idea.Following
- Govindan Ravikumar added an answer:40Nitric Oxide Assay?I would like to quantify NO production from cell lines. I found a couple kits on the market and I am not sure what one to use. Does anyone have any experience using these kits. Can I use colored media?
We can use Griess assay to quantify the amount of Nitric Oxide in Extra and Intra cellular level. But think about NO, it's highly reactive gaseous species it will escape easily (Headspace NO) so Griess assay is not accurate assay. we can use Chemiluminescence based NO analyzer, use air tight HPLC vial and inject both Headspace and solution space and quantify the amount of NO.
DAF always create lot of problem, for this we need proper sample preparation and it's not specific for NO.Following
- Niels Haan added an answer:6Has anyone tried altering the suggested dilutions given in invitrogen Click-iT EdU Imaging Kits?
I have read that (as usual with kits!) the amount used can be significantly dialled down to make the kits go a little further. Does anyone have experience with this?
No idea what the concentration is, but I know it's more concentrated in the final solution than 1 uM, as you get a greenish tinge to the solution at the recommended concentration, but not at 1 uM.Following
- 6Can getting stem cells from fetus harm its growth?
we know that there are stem cells that make our body in our very first months,the stem cells can make different organs of our body and if we have these cells we can help body cure some disease.these stem cells are faster than adult stem cell.but as the fetus needs these stem cells to form the body, isn't it harmful to take these cells?
I agree with researchers .
Look at The ethics of use embryonic stem cells .
Also , These's my two research about stem cells in placenta and umbilical cord of human :
Hope help you . Good LuckFollowing
- Amit Chaurasia added an answer:1embryonic stem cellscan we stoped the aging of cells
This can't be stopped but definitely can be slowed down.Following
- Leontien Bosch added an answer:17Can someone provide tips on culturing iPSc?
Update: Cells are growing fine in our lab now!
I'm the only person in my lab starting on iPSc culture (we received a vial from another lab and all I have is some the medium they use for culture) and I'd like some tips and help. I have human iPS cells, and I culture in mTeSR1 on matrigel coated plates (so feeder free), replacing media daily. I try to remove what I think is differentiation by marking on the plate while looking through a microcscope. One cell line shows nice colonies like I saw on suppliers webpages (Like Stemcell or Invitrogen), the other I'm not so sure...
I've attached some pictures, can someone tell me if this is what it's supposed to look like? And can give someone tips on how to passage? I've passaged one with gently scraping, like i saw mentioned in literature and tried picking the colonies with a p1000 tip (but didn't know if I actually picked up a colony or not, so I tried scraping). Maybe i just need a bit more patience :)
While awesome to start things up, it's frustrating having no one to ask if things are supposed to be this way :)
Thank Sun Yung, I have tried ReLeSR but in my hands it didn't give good results. I'd like to thank everyone for the tips and sharing their knowledge with me, these iPScs are growing quite well now! On to the next steps :)Following
- Mohammad Jafari added an answer:5How do I isolate mesenchymal stem cells from peripheral blood?
I wanted to know if anyone had experience isolating mesenchymal stem cells from peripheral blood.
And possibly what method do you recommend.
Thanks to all
Answers were very useful and practical.Following
- Young Daniel Cho added an answer:10Why do my hBMSC not adhere to the culture dish? What are the possible reasons?
I thaw two vials of hBMSC which are not labelled date or passage. The cells are also suspended in medium 36 hours later after thaw. The medium is human bone marrow mesenchymal stem cell growth medium from CEFO. The composition of the medium are 90% basal medium, 10% supplements and 0.5% streptomycin. Ten days ago I culture another vial of hBMSC with this medium, the cells adhere to dish 24h later after thaw and grow well. So, why the cells don't adhere to dish this time and what are the possible reasons? Thank you!
In my opinion, it's critical to work efficiently and quickly. When thawing cells in DMSO, I simply immerse the vial in warm water just long enough to form a liquid barrier around the frozen "core". I then take an empty 15 mL conical, place the freezer vial upside down in the opening and hold both in place with one hand. I then utilize a "flicking" motion to drop the still frozen core into the vial and quickly immerse in 5 mL of pre-warmed complete media. Basically, the same thing you are doing except on a shorter timeline. This minimizes the damage to the cells.
Keep in mind that if the process of putting cells into the DMSO media is also slow and inefficient, your cells may become unviable before you even freeze them. So perhaps these particular samples were not handled efficiently?Following
- Milena Doroszko added an answer:21In MTT assay after drug treatment do we incubate the cells in fresh media and then check the viability with MTT?Or do we directly add MTT in the drug treated wells?
i wouldn't wash cells on 96well plate since they don't detach (not saying it's wrong, but I prefer not to interfere) but definitely I would dissolve the reagent in your fresh culture media w/o phenol red and antibiotics Before the incubation. MTS assay is indeed easier but it's more of a viability test than proliferation (MTT sometimes is treated as a proliferation test- I don't agree on that but it's very cheap and as a tool before checking apoptosis etc. Is good enough). So depending on what you want to prove You should pick the test. cCk-8 test has a really broad range of detection, so that would be definitely a MTT test I would go for. If you're searching for an MTS kit, I'd reccommend Promega's kit i used many times.Following
- David Jones added an answer:6Any suggestions for a buffered media to run a 16 hour cell experiment without carbon dioxide?
I am setting up an overnight live imaging experiment on our microscope. I have a chamber which I can regulate at 37C and maintain humidity. What I don't have is carbon dioxide regulation. I will be using mesenchymal stem cells and the experiment runs for 16 hours. Will a HEPES based media be sufficient to buffer my media from carbonic acid build up
We have used HEPES successfully for these types of experiments, but added 1/10 the usual amount of sodium bicarbonate. This maintains intracellular pH otherwise the cell drifts into acidic. Cell growth of MSCs is OK int his medium for a week.Following
- Lei Shang added an answer:10What is the important time point in mesenchymal stem cell differentiation?
Hello, everyone. One thing I just want to ask you, because I am a freshman in bone engineering.
We all know that Mesenchymal stem cell could differentiate into osteocyte. In this process, Mesenchymal stem cell may first differentiate into osteoprogenitor, and then into pre-osteoblast, then into osteoblast, finally into osteocyte.
My question may state as following, "what is the important time point in Mesenchymal stem cell differentiation in every differentiation stage??" For instance, when Mesenchymal stem cell differentiate into osteoprogenitor or pre-osteoblast or osteoblast or osteocyte??
Thanks a lot.
Thanks for all the answers above!!
I have not do some research at this field. But Thanks for all the helps.Following
- Gustavo Amorim added an answer:10What is the minimum methanol concentration that can be used as control for tissue culture?I am testing my crude plant extract. It is dissolved in methanol so I am taking methanol as a control. I am using 100ul for 2 ml media.
Dear Daniela ! Thank you so much for your clear, straight forward explanation of methanol usage. I am just about to star my experiment and I have tried diluting Beta sitosterol - Stock Solution - in many different concentrations of DMSO and or DMSO / Ethanol. I will proceed with methanol (respecting the ranges you have mentioned) with an information that really made my day !
All the best success for you and God bless you
Peace and Health
RMIT Uni Melbourne / UFES BrazilFollowing
- Atakan Ekiz added an answer:9Re-use Miltenyi columns?Does anyone have a protocol for reusing Miltenyi columns? I heard that it is doable and popular but just could not find a detailed protocol for that. thanks.
Marc Jenkins' lab mentions using them more than 10 times. As far as sterility... Rinsing used columns with EtOH before re-use didn't give me any contamination problems in in vitro setting. Out of curiosity, I also tried autoclaving the columns. Plastic didn't melt and although I haven't tried using autoclaved columns, I don't think magnetic properties would be affected. Something to keep in mind if extra sterility is needed.Following
- Natalia Oddone added an answer:5What conditions do you use to induce a breast tumor in Nude/J mice with MDA-MB-231 cells?
I injected 1 x 106 MDA-MB-231 cells into mammary fat pad of Nude/J mice. It has been 22 days of the injection and the tumor did not developed.
Any suggestion? Thanks
It wasn`t necessary to use matrigel. Luckily tumors developed by injecting 4 million cells intramammary. Thanks for your suggestions that were very useful.Following
- Kannan Thandavan Arthanari added an answer:13I'm facing problem isolating mouse (C57Bl/6) adipose tissue MSC, while using collaginase 1A according the protocole used in litreasures. Need help!The protocol used by the lab:
1. Dissect adipose tissue from subcutaneous site and cut into small pieces.
2. Digest in 1mg/ml collagenase IA for 1 hour at 37 ºC with shaking 200 rpm.
3. The released cells will be centrifuged at 400 X for 15 minutes.
4. Suspend the pellet in PBS 1X and filter through 70 µm mesh.
5. Centrifuge for 5 minutes at 1500 rpm to wash the cells.
6. Decant the supernatant which contains the adipocytes.
7. Suspend the pellet in BD pharm lyse Lysing Buffer to lyse RBCs.
8. Wash the cells by centrifugation with PBS 1X 1200 rpm -7 min – brake 3.
9. Suspend the pellet in 1 ml Stem X vivo media.
10. Test the cell viability and number by trepan blue on hemocytometer.
In my lab experience Type-I Collagenase gave good results. But I use
to avoid using Lysing Buffer. Sometimes it may damage the other cells too.
Dissolve the Collagenase in plain DMEM and the cells
can be handled in DMEM. Use of cooling centrifuge will give
good result. Cells can be transferred into DMEM + FBS during seeding
in culture flasks.Following
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