- Vijay Saxena added an answer:10How 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.
Try adding more of Lysis solution and I would also ask you to take the reading in nanodrop after allowing your RNA to stabilize and dissolve uniformly in elution bufferFollowing
- Niels Haan added an answer:3Has 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?
Their fluorophore concentration is ridiculously high in the protocol. When making my own EdU staining reagents, I used 1 uM azide-488. Haven't tried this with the kit though.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
- Sun Yung added an answer:16Can someone provide tips on culturing iPSc?
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 :)
A neat trick to clean up your culture without the need to mechanically removing the differentiated cells (second image) is to us Stem Cell Technologies' ReLeSR:
Very easy to use and you will find that your colonies will improve with each passage and differentiated cells will remain attached to the old plate.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
- Damien G Harkin added an answer:3From where can I get contact lens for rabbit?
I want to use a contact lens for rabbit for transplantation of stem cells to the corneal surface with substrate. i want to put a contact lens which can also able to diffuse gas (oxygen and CO2). I want to use this lens just to avoid the problems due to blinking. Please suggest me from where I can get this. Please also suggest me, what other methods would be possible. As I am also not able to suture the cell-substrate sheet. I also would like to get information about other ways to put the cell-substrate patch over the cornea other than chemically defined bioadhesive. Please do help in this. Thanks!!!
If nictitans is a problem, you can suture across the top of the implanted test material. Vets treating epithelial defects in cats and dogs often perform this.Following
- Wulligundam Praveen added an answer:5What is the importance of high no. of passages in characterization of pluripotent stem cell lines?
I would like to know the possible upper limit of no. of passages for pluripotent stem cells.
Dear Clara, Jorg and Ajay,
thank you very much for the valuable inputs.
- Dina Hazem Gomaa added an answer:10How can I interpret scanning electron microscope photos of salivary gland stem cells?
My PhD thesis title is "isolation and charecterization of submandibular and parotid salivary glands stem cells of albino rats". And one of the investigations i used was scanning electon microscope. So i have photos of salivary glands stem cells but cant write comments. I searched alot and cant find any related articles. Would appreciate it if i can get any help. Thanks a lot
Dear Dr Yash,
thanks a lot for your concern and reply, really appreciate itFollowing
- Maria Goes added an answer:8Does anybody have problems using ALDEFLUOR assay buffer?
I am trying to perform and aldefluor assay but whenever I use the buffer, the FSCxSSC scatter gets messed up and a great part of my population dies.
But if I run the same cells on PBS the scatter is perfectly normal... Is there any other alternative to using this buffer ?
Thats the point, I am performing the same centrifugation with other cells in PBS and they don't die - their scatter is just fine . In the beggining I was performing a slower centrifugation (300g) but I was having very few events - sometimes 10% of the cells I had originally + bizarre scatter. Now I have about 50% yield. I wonder if it could be because I am centrifuging them at 4oC, right after they left the 37oC incubation. But, again, the other half in PBS survives this just fine.
I might email the sales representative with the bizarre scatters and ask for a solution though.. sounds like a good plan !Following
- Augustine Thambaiah added an answer:6Is it feasible to obtain 100 Million Mesenchymal Stem Cell in a 2 Layered Cell Factory?
I would be using 2 layered cell factory (1272cm2) for Mesenchymal stem cell culture.
Source - P1 Umbilical cord Mesenchymal Stem Cell.
If it is feasible to culture and arrive at the magical number (I100 Million mesenchymal stem cell ). what would be the ideal media to use, Supplement ( FBS % and bFGF concentration ) and Seeding density ?
70-80% confluent flask (any type) will have ~20,000 - 30,000 cells/sq.cm. if you calculate based on this a two layer cell factory (~1260 sq cm) should not have more than 40 million msc at 70-80% confluence. and the recovery of cells from the flask by trypsinization will not be 100% you will still have some cells left in the cell factory. recovery will be around 80-90%. so the final yield from a two layer cell factory will be 30-35 million msc.Following
- Nick Asbrock added an answer:9How do you analyse FACS data obtained by the ALDEFLUOR kit?How do you analyse the FACS data obtained by the ALDEFLUOR kit? Where do you set the gate on the inhibited sample (DEAB)? What should be the cut-off on the inhibited sample? If you set the ALDHhi gate really close to the cell popluation in the DEAB sample then you have a rather high intra-experimental variation.
Look forward to hear from anybody who worked with this kit.
EMD Millipore has recently commercialized a red-shifted fluorescent substrate for aldehyde dehydrogenase (ALDH) that works in a similar fashion as ALDEFLUOR but allows the concurrent use of green fluorescent cell lines, antibodies, transgenic animals and reporter assays.
Attached is the Nature publication describing the AldeRed reagentFollowing
- Balasubramani pk added an answer:12Are collagenase IV (gibco) and Hyaluronidase (sigma) harmful to cells?I digested umbilical cord tissue with collagenaseIV(0.5mg/ml) and Hyaluronidase(0.1mg/ml) for 12h, but after digestion very small number of cells were collected, and it took 2 weeks to form MSCs colonies. In many papers , the MSCs colonies were observed after several days, and collagenase I or II are usually used. So, are the two enzymes harmful to cells?
If my starting material (Umbilical cord is 5cm in length) and both artery and vein removed from the tissue !! What would be ideal volume of C & H added ? Please specify the concentration of the C & H enzyme too?Following
- MOHD AQDAS added an answer:19Cells remain adherent irrespective of trypsin treatment?I am working on Mesenchymal stem cells derived from murine bone marrow. One thing I have noticed that MSCs when trypsinized remain adhered and trypsinized for 8 minutes. The cells which float are unable to adhere in plates after subculturing.
I use TPEG solution (0.2% trypsin; 0.02% EDTA; 0.05% glucose in PBS) for trypsinization.
Why I get a white clump after centrifuging the trypsinized mesenchymal stem cells?? is there a way to get rid of it?Following
- Ashaq husssain Najar added an answer:6Neurons obtained from Human Neural Stem Cells and grown for ~ 45 days failed to fire when patched. Could anybody advise how to solve the problem?
I have generated NSCs from many human pluripotent stem cells (Human ES and iPS lines). I am trying to differentiate these NSCs into pan neuronal populations in Neurobasal medium supplemented with B27 (with Vitamin A) and Glutamax. On day 6-7 of differentiation, I add cAMP for 3 days. Then cells are grown in the above differentiation medium for upto 45 days till the cells show fine neuronal morphology. These cells get somewhat stressed over time. They stain positive for early and mature neuronal markers but when patched, fail to fire. I use PDL/laminin-coated glass cover slips for growing the cells on. Are these cells not mature enough to fire? what could be the other reason/s for not getting functional neurons?
Thanks a lot Adrian. I am NOT using either of the factors BDNF /GDNF. Just basal medium, B27 supplement and cAMP. You are right in saying that electrophysiology of these cells is very much dependent on the type of media used.Following
- Roberto Ensenat-Waser added an answer:6What is the best and the most simple protocol for Embryonic stem cells karyotyping?
I'm doing karyotyping in mouse embryonic stem cells,but it is really difficult to count chromosome number accurately, requiring a long time to find good metaphases.
I would like to know if someone could help me with a better way to spread chromosomes, and provide some tips to facilitate counting.
Thanks a lot!
As Saleh said it helps adding colcemid or colchine, but normaly with mESC growing at their normal rate you should get plenty metaphases. In my hands the critical steps was the height and even more aiming properly the drop on the coverslip. use the position of your hand in respect to your head as reference. you will need a couple of tries to get the proper one, then measure it andn write it down.
another trick, put the coverslips on dry ice to ensure they're really cold.
For the counting you have to be really patient and be realistic with the acceptable n, I was doing fusion between ESC and SSC and we realised that at least 15 are a must but between 20 and 40 should do the job.
Finally check if you have a medical faculty or hospital around, if big enough they have Karyotyping facilities and setting the software to count mouse chromosomes won't be an issue. They may ask you, though, to have the cells ready and fixed.
- Saleh Alkarim added an answer:4Is there any chemokine that responds only to stem cells?
Are there any chemokine receptors found only on stem cells and not on mature leukocytes?
I agree with all researchers , see the links here i hop help you
- Tomo Saric added an answer:4Has anyone had any problems while using dispase to split iPSCs?
We are been having trouble lately when using dispase for more than three minutes. Our feeder cells are coming out with our colonies in the wash step as a one layered mess. When we re-plate our cells to expand, there are this ugly strings of MEF cells all over the plate. We thought it was mycoplasm but our culture is negative for it. We also tried to dilute our dispase (StemCell @ 5U/ml) and change the incubation time from 5 minutes to 3 minutes but with no luck. Could it be the gelatin we use when plating the MEFs? We have looked at the MEF plates before using to plate our iPSCs and it look fine. Any thoughts?
We haven't use dispase for splitting our human iPSCs but have very good experience with EDTA (i.e. Versene) for splitting iPSCs grown on vitronectin-coated plates and cultured in E8 medium. This is a gentle, enzyme-free and time-saving procedure that allows both the stable passaging of iPSCs adapted to feeder-free conditions and their efficient cryopreservation. If you continue having problems with dispase you may consider switching to EDTA procedure because it will also reduce your costs associated with generation or purchase of MEFs, which itself may unnecessarily introduce variability in your iPSC cultures.Following
- Wen Li added an answer:3Does anyone have good experience to convert human ESC to naive state by 3iL(Ng) or 4i(Hanna)?I m suffering a little trouble ...?
I used two cell lines hESC cutured in E8 medium feeder-free condition.When I convert into naive state use protocal by 3iL or NHSM media,I find the colonies shows continuous apoptosis and cannot passage even though forming a little dense colonies as picture.First I think the high concentration of every inhibitor harms to cells in consideration of individual cell line,but when I reduce the dose,it does not work.
Does anyone have similar experience or can give me advices?
Only some perspective according to current knowlegde. I have no experience on coverting primed hESCs to their naive state. If primed hESCs represent epibalst stage, depending on TGFb/FGF2 signaling to maintain their self-renewal, while naive hESCs rely on 4I or 3i, representing earlier stage compared with epiblast stage. It might not so easy to convert the later stage cells into early stage by just changing the culture condition. The success report on that simple way might be some remained naive stage cells within the primed culture, which can survived in the 4I or 3i environment and be enriched because primed cells die out just like what you observed. I remember that some pluripotent gene, KLF4or KLF2, when ectopically enhanced in primed hESCs, is sufficient to push them back to the naive state, by both X activation and TGFb/FGF2 independent.Following
- Juan Uriel Mascotte Cruz added an answer:7Does anyone have any experience with in vitro differentiation of neural stem cells at various plating densities?
Anyone have any experience with in vitro differentiation of neural stem cells at various plating densities? I have found that plating at densities within the usual recommended range (50-100,000 cells/cm2) does not give an adequate amount of RNA from TRIzol extraction.
However, from what I understand, too high of a density may inhibit differentiation. I am planning on trying 100-500,000 cells/cm2. What upper limit have you found, if any, where neural stem cells are unable to differentiate as effectively?
As Pavel Ostasov said, I commonly seed 300,000 cells/cm^2 and they grow well, but if you want an extraction, You can wait until the first 4-5 days. And what was your medium? Because you can add different concentration of FBS and this is going to modified your final concentration of your cells for the extraction. All in a flask of 75cm^2! good luck!Following
- Jaya Aseervatham added an answer:17Can I loose the protein of interest from a stable clone?
Iam facing a weird problem after transfection. I am using C3H10 meschencymal cell line which is a stable clone expressing a protein which is GFP tagged. Iam using this cell line to transfect another protein having a red tag. both the protein has G418 resistance. After a month, I have few colonies. But when I see it under the fluorescent microscope the resistant cells neither have GFP or red tag.
1 . How is this possible, when I have used a stable clone with a GFP protein ( I did check the cell for fluorescence before the experiment.) for transfection.
2. Do the cells loose the protein for any reason.
Suggestions are welcome
Thank you Patel. I will discuss it with my PIFollowing
- Jessica Bowman added an answer:4Is anyone able to help with immunocytochemistry before plastic embedding?
I'm currently trying to optimize a protocol for plastic embedding and sectioning human embryoid bodies (EBs) from H9 stem cells. Since I am trying to show that our labs way of developing embryoid bodies is potentially more organized in shape and growth compared to other previous methods for making EBs, I wish to show the 3 germ layer formation, ideally through ICC before embedding and sectioning.
I am looking at this kit from R&D http://www.rndsystems.com/Products/sc022/
but I know there are some varying levels of success with antibodies and plastic embedding...
Has anyone previously used the antibodies that come with this kit, or the kit itself with plastic embedding? If so, what were the downfalls? Better success with ICC before or after embedding?
Basically, any info on these antibodies with use in plastic embedding would be much appreciated!
Hi all, thank-you for your answers. There's a bit more confidence added to how I will go about this.Following
- Budd A Tucker added an answer:3Does anyone have experience with amaxa 4D system for nucleofection of human neural stem cell?
I have to nucleofect 2 cell types. I have access to amaxa 4D system. Standard transfection with lipofectamine 3000 and Mirus Lt-1, I get approximately 30-40 efficiency, but most of thetransfected cells die.
1) iPS cells
2) iPS derived neural stem cells.
Does anyone have experience with amaxa 4D system? Conditions, cell number, plasmid quantity and program to use?
Thanks a lot!
We have used the 4D system quit a bit for hIPSCs, we use the P3 kit with program CB150 in either 25ul strips or 100ul snap top cuvettes. The key is to plate the cells back post-transfection at a rather high density, i.e. ~1x10^6 cells per well of a 24 well culture plate. George Church has a good paper describing the technique in detail for CRISPR work, DOI: 10.1002/0471142727.mb3101s107.
As for human neural stem cells we have done photoreceptor precursor cells with similar methods, there is a rather useful optimization program that is built into the 4D system that would help you optimize your protocol. For human photoreceptor precursor cells the P2 kit and program DS150 works well (~70% efficiency).
Hope this helps.
- Hasan Serdar Mutlu added an answer:3CM-Dil Cell Tracking for HSCI tried to stain Hematopoietic Stem Cells with Cm-Dil fluorescent carbocyanite according to the providers manual for cells in suspension. I stained 3 min at 37°C with the working solution (1µM) and further stained at 4°C for 15 min. Then centrifuged, washed in PBS 1x and resuspended in media again. The staining did not work :-)
The cells were not freshly isolated but already 5 days cultured in StemSpan media....
Has anybody experience with HSC in this context?
What color did you see CM DiI in your sections?. I labeled cells with CM-DiI 1µM concentrations 5 min. at 37 C and 15 min. 4 C. And then injected in vivo. After 5 days from injection, I saw greenish florescent in formalin fixed paraffin embedded sections. Is it correct ????Following
- André Luiz Lourenço added an answer:6How can I measure HUVEC express P-selectin and a derivative/P-selectin interaction?
I'm starting to work with HUVEC and several works do this assay with HUVEC, but the majority induces in different manners.
I would seriously recommend using fluorescence for the detection of P-selectin expression. BioLegend provides a plethora of anti-human CD62P (P-selectin) with fluorophores. You can use the common FITC or AlexaFluor647-antihuman CD62P antibodies to mark P-selectin in live cells and quantitate the signal using flow citometry or confocal microscopy.
Alternatively you could coat microplates with collagen or fibrinogen (purchase a Maxsorp 96-well flat bottom microplate, add 50 microliters of a collagen/fibrinogen concentrate and incubate overnight at -4C (adding a surplux of BSA is advised). In the next day you should have a coated microplate with collagen or fibrinogen located at the bottom. From here you could add platelets (isolated or directly from PRP)...wait for 15 minutes and perform washing steps. After the washing procedure you'll be able to quatiate platelets/HUVECS that are still bind to the coating-cell network in each well through FITC-anti-CD62P antibody.
If antibodies are not available and you need quick results, you can always try -FITC-phalloidin. And if phalloidin is also hard to find for any reason, you can stick to the traditional approach by using Giemsa staining (Check publication attached below).
I'm adding a thesis from Andreas Eriksson which covers a lot about ways to assess platelet adhesion and function using highthroughput microplate assays, which may also work with HUVECs if you choose the right matrix.
Hope that helps.
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