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Stem Cell Isolation - Science method

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Anyone have experience using Stem Cell's Sepmate PBMC isolation tubes with LEUOKOPAKS (as opposed to whole blood)? Are Sepmate tubes compatible with leukopak preparations, or only with whole blood? Thanks.
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Chiming in (years after the initial convo lol) since I used SepMate extensively in my lab days and now work for STEMCELL, and we get this question all the time.
Officially, SepMate is only designed to work with whole blood and bone marrow, because the vials require a minimum packed volume of red blood cells in the pellet to keep the MNCs from getting trapped below the plastic insert. Leukapheresed blood has many fewer RBCs than whole blood, so the RBC pellet is too small.
However, unofficially, many labs do use SepMate with leukopaks, and compensate for the low RBC count by adding an extra 3 mLs of Lymphoprep/Ficoll to a 50 mL SepMate vial to keep the MNCs above the insert. You will have more mixing of the cells and density media this way, so pour on the diluted leukopak contents more slowly than you normally would. You can still spin for 10min at 1200g with brake on per the protocol. Obviously test it with your own setup (STEMCELL gives free SepMate samples) but, yes, it is a possibility.
Hope that helps any future researchers googling this question!
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Hi everyone,
I have the problem in Hair Follicle Stem Cell Isolation. Could people kindly help me, please ? Thank you so much!
I am trying to isolate the Hair Follicle Stem Cell and Keratinocyte from Mice Dorsal Skin. Here is our brief protocol :
+ The dorsal skin was incubated in Trypsin 0.125 %, 2h30 at 37C
+ Put skin in cold PBS + 10% FBS to prevent enzyme activity
+ Scrape all hair off the skin using forceps and scalpel
+ Break down the Hair Follicles using scalpel, forceps, and pipetting 10 times
+ Filter 70um and 40um, then centrifuge 300g, 10 mins
+ Resuspend in Keratinocyte Media ( low Ca2+) and centrifuge 300g, 5 mins
+ Acquire pellet and count cells.
However, the viability is just around 30-40 %. And when I seed cell, the adhesion is low ( I used gelation for coating). After I change media, a lot of cell removed.
So could people kindly give me any advice to increase the viability as well as the adhesion ?
I truly appreciate your support and have a nice day.
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Dear Kai,
First of all, I truly appreciate your kind. It support me a lot.
I would like to isolate the hair at telogen stage, so I use mice 6-7 weeks old.
I tried to incubate with Trypsin ( 0.25% and 0.05 %) overnight, but the viability was also around 30%-40%. So whether if do you know normally how is percentage of alive cell isolated from dorsal skin ?
Regarding cell adhesion, I have just changed to use media added FBS or Serum Replacement, the adhesion is improved. I am just wonder the Calcium concentration in Serum will induce the differentiation or not.
Thank you Kai very much and wish you all the best !
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We isolate NK cells using a kit from Stem Cell Technologies but since the proportion in PBMC is relatively low we're looking for a method to enrich them before using the kit.  Has anyone used this or another method that might be helpful?
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I would like to isolate NK cells using your mentioned technique and the RosetteSep Isolation Kit from 25 ml whole blood. So could you guide me on the details of the protocol of how you isolated the PBMCs (speed, ratio) and if any dilution was done? Also, if I would like to check the purity for NK cells what is the best marker that I should go for ?
Thanks so much for your help in advance.
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Pericyte and SMC have same marker and functions. I can't find any publications to differentiate pericyte from SMC?
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This could depend on the tissue you are examining (if it is tissue). 
If you wanted to identify brain or skeletal muscle pericytes then this simple technique could be useful.
Microvasc Res. 2013 Sep;89:164-8. doi: 10.1016/j.mvr.2013.05.008. Epub 2013 Jun 10.
A simple method to fluorescently label pericytes in the CNS and skeletal muscle.
Edwards IJ1, Singh M, Morris S, Osborne L, Le Ruez T, Fuad M, Deuchars SA, Deuchars J.
PMID: 23764127
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Hi I would like to isolate CD34- (negative) cells from cord blood and culture them? Can anybody share their experience with isolation and culturing?
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Hi All, I am trying to isolate neural stem cell from mouse embryo E13.5 forebrain. After that, we perform magnetic bead assay to purify neural stem cell population but after doing purification stem cell NSCs are not forming neurosphere. Please help me regarding this with your experiences. Thank you.
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Control culture? I am not getting this.could you please elaborate?
We used SSEA1 antibody.
Thank you
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which tissue is beter for isolation of MSC from Adipo? and how mach we needed for getting stem cell clone ?
best culture media for culture of human AD-MSC?
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Hi,
you can isolate Ad MSC from human lipoaspirate or from subcutaneous fat from abdominoplasty surgery, for example. Both protocol are quite different.  Most common is from the lipoaspirate. You will need to wash the lipoaspirate with PBS or saline solution to take out blood cells. You can do that using a separating funnel. After that, you incubate the lipoaspirate with colagenase IV for 30-45 minutes at 37ºC. Then, you block the colagenase with Fetal Bovine Serum and centrifugate at 300G for 5 minutes. You can use a red blood cells lysis solution at this point . After that you suspend the pellet in DMEM F12 and filter the solution with a 70 micrometers cell strainer direct on a culture plate. You can mantain the cells in DMEM F12 + 10% Fetal Bovine Serum. Its a simple protocol. Good luck.
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I want to detect BDNF in conditioned media derived from MSC(bone marrow, adipose tissue) by ELISA. I found in articles that it is needed to be concentrated(approx. 40x) at cells density approx. 5.106 /10 ml media. Does anyone have an experience with it? If I concentrate the media do I need to concentrate media for control (blank)? Do I have to do the samples dilution afterwards?  Thank you.
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Thank you very much.
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Hey there. I need to isolate stem cells from mouse fetal membrane, for a project I'm working on. but I know nearly nothing about it, only a little about mating a male and female mice and pregnancy duration! but exactly how and when can I extract fetal membrane from pregnant mice and could it be after breeding the babies? I hope it would.
waiting.thanks a lot.
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tnx a lot!
sorry for  answer back this late, there was a problem with my connection
regards, Sara Vahdat
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This is the positive fraction. I don't know if it's contamination or just debris of dead cells
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Looks like contamination.
Even if it's "just" debris from dead cells there are some issues in your samples. Why should u lose so many cells and end up with dead cells being your major compartement?
Debris of dead cells often cause significant changes in the remaining cells and my cause your rest of the culture to die too.
You should repeat your isolation and investigate the cause before, imho.
Best regards
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Hi,
I have successfully isolated stem cells from fresh urine but I am trying to figure out if the urine sample can be refrigerated for a period of time equivalent to shipping on ice and still get cells out. I am setting up an experiment to try and isolate cells from urine that is refrigerated for 24, 48 and 72 hours but I was wondering if people have done this. I haven't found many papers online. Any suggestions are welcome.
Thanks!
Elisa
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Hi Elisa, you could consider freezing them at-80oC in the presence of DMSO.
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Hi ,
There are a lot of protocols but most of them requires culture these cells as a part of the technique , for my project i need easy protocol to preparation and analysis hMSC of placenta tissues without culture cells ( mean work direct on tissue)
Best Regards
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I agree with Pankaj Mogha. FACS Sorting is your best option. You have to select one or two population before hands in order to buy labeling markers whenever you intend to do sorting. And frankly it will be expensive if you choose to sort and isolate 4 or more of different cell types. In this case culture will be better off for you.
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I read some papers that use G-rex or conventional tissue culture flasks. Which one do you suggest? Which medium is the best? 
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Somanchi, S. S., Senyukov, V. V., Denman, C. J., & Lee, D. A. (2011). Expansion, purification, and functional assessment of human peripheral blood NK cells. JoVE (Journal of Visualized Experiments), (48), e2540-e2540.
I follow protocol from this paper. I use IL 2 along with RPMI and 10% FBS. I keep NK cells suspended in T75 flask in standing position.
Hope this helps.
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Dear colleagues,
I culture mononuclear cells(MNCs), isolated by density gradient centrifugation, from the cord blood of preterm infants. I aim to culture these MNCs into endothelial colony forming cells (ECFCs), on culture surfaces coated with 2% Gelatine from PAN-Biotech.
In many of my samples, I have noticed a progressive reduction in number of MNCs (before they get to ECFC) as I change(refresh) the Endopan3 PAN-Biotech culture medium.
(NB: Before  culture, the freshly isolated MNCs show almost 100% viability, with Trypan Blue)
I will be very grateful for any contributions to why this mishappening occurs and how it can be solved.
Thank you in advance.
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Hello
Yes try with insulin .
Please see this link will help you
Good Luck
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I want to culture spheroids and isolate stem cells from immortalized human proximal tubule cells. I have been able to get single suspension of human proximal tubule and seed them into ultra low attachment flasks but I want to pick one sphere and grow them on a 6-well plate to see if they can produce a clonal population. I am just beginning to do this, I do not have a lot of idea on these type of isolation techniques to obtain a stem cell population, so I would appreciate any comments/ suggestion. Thank you
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Dear Mai
Great, I wish you a lot of success! If you need assistance, please let me know.
Regards,
Patrick
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There is a PLOSone paper indicating that it did not work very well.
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Hi, could you possibly provide that PLOSone paper? I'm also deciding between MACs and EasySep and wanted to hear all arguments before deciding. 
Thanks 
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Hi! I wanted to generate Embryoid bodies(EB's) from mouse embryonic stem cells in a non-Tissue culture treated flask. Now I need to transfer them to a tissue culture treated flask for directed differentiation to neural lineage. I would like to know the protocol for passaging them. Can I use the centrifuge to get the pellet, followed by resuspension and then seed them? or should I do gravity centrifugation? The reason for my question is, If I use the centrifuge and resuspend the pellet, they would no longer be in EB form and I dont think they can be used for differentiation? Since this is the first time I'am doing this, I would like to have some suggestions to proceed with this issue!
Thank you!
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Hello there,
I am working to isolate breast cancer stem cells from MDA-MB-231 using CD44+/CD24low positive selection on magnetic separation (Miltenyi Biotech column). Has anybody used this kit for solid tumor cells and not hematopoietic cells that the protocol is based on? I reach a good enrichment but I end with very low number of cells to work with. Any suggestion is helpful.
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As far as i know, almost all MDA-MB-231 cells are CD44+/CD24low or negative. I would suggest to perform flow cytometry before and after purification. A caution: If you are detaching cells with extensive trypsinization, then the cells may loose certain surface markers (potentially CD44 as well).
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I tried to isolate HSCs from two old male C57BL/6 mice. after nycodenz step, i didn't get white layer of HSCs even overlay of GBSS/B is clearly visible. when i culture upper layer or pellet, i got very less amount of cells with lot of debris and is it necessary to use protease also with collagenase during digestion step? please give your suggestions. i follow attached protocol.
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Hello saleh.
Hepatic stellate cells may start to proliferate so they could be called as progenitor cells but these are not stem cells and their number increases with age. Thanks you
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Hi, I only worked with bacteria so far and recently changed fields. I am now working with stem cells. I find it difficult to adapt to new protocols. Unfortunately, I can't say I'm getting enough help in the lab. What are the best resources I can use? I really need to get up to speed with these new methods. Although I'm reading paper after paper, I don't think it's fast enough. Thanks a lot for your help. 
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Dear Ilkay Gulsoy,
Your question requires a very broad answer but to be very precise, I would suggest you to start doing something practical in your lab and you will find the world of stem cells really beautiful. Reading many protocols at a time will only lead to confusion. Read 1-2 protocols at a time and try to apply them practically. I would also suggest reading some very good blogs like Knoepfler's lab and CIRM. These will make you friendly with latest advancements in the field as well as help in developing your interest in the area (as they narrate some very exciting stories sometimes). For a good knowledge on  stem cell culture, please go through R. IAN FRASHNEY's book "culture of human stem cells" by Wiley publishers. This will help you a lot in clearing basic concepts and problems encountered in routine stem cell culture.
Please feel free to write and ask in future about anything related to stem cell field.
Hope it helps !
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What collagenase enzyme should be used to isolate human adipose derived stem cells and what concentration should be use on liposuction material?
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Hello
Adipose derived stem cells :
Collagenase Type 1: 0.15%
Collagenase Type 2: 0.1%
for more optionis see this links will help
Good Luck
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I have isolated neurospheres from a neuroblastoma cell line, passaged them once and they seem all right for the time being. I will also do the same experiments from NSCs isolated from adult mice. However, I have to do overexpression and downexpression experiments with the isolates. We only have PEI and I am not sure about the efficiency of the transfection. As there are not many cells that I can give up for the optimization experiments, I am trying to find out a strict protocol. I know NSC transfection is though. Any recommendations and protocols would be appreciated.
Thanks in advance,
Melis 
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Regarding fixing the cells 
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4% PFA is fine but make sure it is freshly prepared. Rinse your cell line with 1X PBS before fixation. Regarding lost of cells, I think you should optimise your washing technique. Do it as gently as possible. 
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I have been trying to isolate mesenchymal stem cells from umbilical cord. I followed both explant and enzymatic digestion techniques. Few cells were found in digested culture after 2-3 days; however, no proliferation was found. In explant culture, unlike RBCs, I observed spherical shaped cells around the small pieces of cord tissue. After 7-10 day cells started to lyse in both culture. I used DMEM with 15% FBS (US origin) and 1% pen-step as complete culture media. Can anyone suggest me how would i overcome the problem?
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Please suggest me standard Alpha-MEM composition used for MSC culture. What type of alpha-MEM (with nuclesides or without nucleosides) should I use?
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We have an EDC crosslinked collagen based material and are performing a degradation assay to understand the byproducts released and quantify them. Is there an assay that exists to detect EDC in any form (residual, unreacted, partially reacted, fully reacted) as the material degrades or upon initial hydration? I understand there is a free Chlorine when EDC is unreacted that is no longer there upon successful crosslinking. Our initial thought is to focus on the presence of Chlorine as an indication of residual EDC. Any thoughts are appreciated!
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My pleasure. Hope it works for you and good luck for your submission. 
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I am trying to use Percoll gradients to enrich for progenitor cells isolated from organs. The cells can be dissociated and are viable afterwards. I am using Percoll Plus from GE Healthcare.  I have been using the protocol in the attached link.  If I follow the protocol exactly, I am able to isolate cells at the different density interfaces, however, all the cells are dead when counted using trypan blue exclusion and a hemocytometer. I found some other protocols that use 400 x g for centrifugation and I have set different ranges of RCF from 200 to 600 times g. In every case, the cells have been permeable to trypan blue. I will try a ficoll underlay and I am thinking about Nicodenz. Has anyone else had this issue? How did you overcome it? Any feedback is welcome. TIA.
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Hi,
We use 10x PBS without HEPES to make up the percoll, then use1x1640(with 10% FBS) to make the gradient to isolate immune cells.
And did you get rid of erythrocytes?
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Hi
Could you throw some light on the best way to perform neurosphere assay (limiting dilution) for neural stem cells isolated from hESc/iPSC? I am looking at the role of a Ca-signaling gene in proliferation of NSC. My NSCs are transduced with shRNA against this gene using lentiviral vectors. I also have the scrambled/NTC as control. TIA
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I'm agree with the colleague. The substrate is the key of neurospheres production! Not necessarily with addition of NSC medium, but the cells need their potentiality for differentiation in neurons.
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I am aware of Streptavidin magnetic beads which can be used with biotinylated antibody in MACS. But I would like to know if anyone has tried isolating or depleting cells from heterogeneous population by labeling cells with purified antibody followed by use of protein A magnetic beads. 
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Fantastic John. BACS was something i did not really know about. First I will answer your query. I am aware of Streptavidin-biotin system and am using it, but cannot go beyond a round of positive selection. Say I want to isolate double positive cells for two antigens. FACS is an option, but I want to bypass FACS because of technicalities with using the machine, amount of time and labour invested, and at times the availability of sorter.
BACS is very interesting and I feel now I will be able to go upto triple positive. So the idea is to perform one round of positive selection with streptavidin biotin and then use  Protein A beads for second round of positive selection.
I think it will be interesting if you can let me know if you can directly conjugate antibody to your microbubbles.
Best,
Neeraj
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 I know there are three isoforms: CD133/1, CD133/2, and CD133/3 and also the fact that CD133/2 is the isoform that is used for hematopoietic stem cell isolation.
Thank you so much in advance. 
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What precautions one should take to maintain viability of stem cells of adipose tissue, during their isolation
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Hello Nisha,
Completely agree with Amita. In addition to the above suggestions, take care not to centrifuge stem cells for too long during pellet down step of single cell suspension you finally get after digestion and even avoid using high centrifugation speeds. It applies to upscaling your culture also. Generally stem cells settle down very nicely for  2k rpm for 2 minutes. High speed centrifugation for longer time is definitely going to kill cells. Last but not least, don't do harsh pipetting on cells, try to handle them gently like babies. They will respond well to your love and care.
All the best !
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Is there a good kit to isolate stem cells from cancer cell line
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Dear Vijay,
Please read the following:
Cancer Stem Cell Products & Services
ProMab Biotechnologies, Inc. offers a wide line of cancer stem cell products and services including cancer stem cell media, cancer stem cell antibodies, and cancer stem cell markers. Cancer stem cells (CSCs) are hypothesized to reside within the tumor mass cell milieu, and retain certain characteristics of normal stem cells. Cancer stem cells are highly tumorigenic, in contrast to other cancer cells, persist in tumors as a distinct population, and may cause relapse by giving rise to new tumor growth with increased metastatic potential. Recent research indicates cancer stem cells display unique properties and resistance to chemotherapy and radiotherapy. This breakthrough could lead to the development of new tests for early cancer diagnosis, prognostic tests, and innovative therapeutic strategies.
Identification and isolation of cancer stem cells from either tumor cell line spheroids (tumorspheres) in vitro, or in vivo tumors, will provide valuable cell populations in order to study their origin and mechanisms of establishment, maintenance, and importantly the molecular mechanisms responsible for their conversion. ProMab’s various cancer stem cell antibodies, cancer stem cell media, cancer stem cell isolation kits and cancer stem cell markers will therefore provide the researcher with an arsenal for the development of cancer stem cell specific targeted therapies, as a means to increase survival rates amongst cancer patients in general, but especially for those at risk from tumors with high metastatic potential. See below for our cancer stem cell antibodies, cancer stem cell markers, cancer stem cell media, cancer stem cell isolation kits as well as other related products and services. In addition to our cancer stem cell line, we also offer custom monoclonal antibodies and protein expression services.
Isolation of cancer stem cells
Flexible: isolate cells anytime, whenever samples come in
Convenient: separate cells independently of flow sorting facilities
Versatile: large choice of cell surface markers for isolation
Diverse cell types within the tumor environment can play different roles in cancer and tumor progression. For reliable analysis of the different cell types it is crucial to work with pure cell populations as contaminating cells might skew the results.
Be able to assess the role of cancer stem cells in your research model using our MicroBead reagents and kits, which are optimized to ensure a high purity and yield of target cells. MACS®Technology allows you to isolate CSCs anytime, whenever the samples become available.
Scientific poster on breast CSC isolation and gene expression in rare cells
Report by Procoli et al. on CSCs in ovarian tumors
Scientific poster on CSC isolation
Overview of human CSC markers. Click on the image for a larger version. 
Hoping this will be helpful,
Rafik
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myeloma stem cell
isolation
sorting
culture
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Given that multiple myeloma arises due to the monoclonal expansion of abnormal plasma cells, which leads to the production of monoclonal antibody characterized by Bence-Jones protein, IgG, IgA, IgM, which is produced in a mutually exclusive manner depending on a clinical case. That is why the typical concept of cancer stem cells in the heterogeneous tumor cell society is difficult to find in multiple myeloma. 
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i want to isolate breast cancer stem cells from breast cancer cell line with purpose of drug researches. can anyone suggest me which cell line is better to isolate?  is it possible for any kinds of breast cancer cell line?
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The following breast cancer cell lines can be raised as suitable for the isolation of cancer stem cells (CSCs); SKBR-3 (luminal), T47D (luminal)MDA-MB231 (basal). THe mot typical method for CSC isolation is the flow cytometry with CD24 and CD44 antibodies; CSCs exist in the CD44(high)/CD24(low/negative) subpopulation. You further better check the ALDH1 activity after isolation!!!
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How to evaluate the capacity of certain small molecules during somatic cell nuclear transfer although it may work well during iPS cells reprogramming, considering the size of somatic cells as to oocytes? many HDACi or other epigenetic modifiers do not work well during somatic cell nuclear transfer reprogramming,especially during later development, which have been puzzled me much.  could you talk about the topics with me?
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SCNT is still superior to the iPSCs because the resulted cells from SCNT are totipotenet however iPSCs are pluripotent and might be uncontrollable. Regarding the effectiveness of the SCNT process; it is multi-factorial as it depends not only on epigenetics but also on the recepient oocytes maturation, the donor cell cycle and even the culture condition.
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I am doing a reprogramming process to generate iPS using Doxycycline inducible OSKM polycistronic lentiviral cassette. But just 24 hours after the first treatment all the Dox treated plates (both cancer and non cancer cells) start to die and deattach from the surface while the non treated cells are normal and viable, so I can not keep the process. Is someone who experienced the same situation? Or does someone know why this happens to my cells? 
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It sounds like something in your treatment is somehow killing them. I would've guessed that the lentiviral particles were the source of the toxicity (in which case, you could incubate them for less time), but your transduced, non-treated control cells are fine.  
I suppose I can speculate. When you prepare your doxycycline dilution, are you filter-sterilizing it before treatment?
I've attached the Mouse STEMCCA Dox-Inducible Polycistronic (OKSM) Lentivirus Reprogramming Kit protocol from Millipore.
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Hi everyone,
I have been trying to isolate ESCs from blastoderm of stage ten chick embryo. I have been trying to remove the yolk and vitelline membrane from the pellet to obtain ESCs with centrifuging specs of 1500 RPM at 5 minutes at 10 degrees celcius and acc/dcc at 22. I couldnt isolate the yolk from the pellet even after repeating the process 7 times. If I were to repeat the process an umpteen number of times, I would have dead cells. Is there a technique that was found to be successful in isolating the ESCs from the vitelline membrane and yolk?
Kasi
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I  want to isolate mesenchymal stem cell from human adipose tissue
which type of media use DMEM or DMEM-f12
and depend on what i choose my culture media for stem cell 
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Hello
DMEM H/L - G and DMEM/F12-based media and both used in the literature for cultures MSC . Also , the DMEM/F12 had established containing stable L-glutamine dipeptide as the optimal basal medium for propagation the MSC .
From me , was cultuerd these cells with adipogenic media containing DMEM low glucose, 10% FBS, 0.5 mM isobutyl-methylxanthine , 1 µM dexamethasone , 10 µM insulin, 200 µM indomethacin , and 1% ABAM.
Good Luck
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Relation grams/n of cells?
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I believe that we are working with the same fat type, with different semantic words, why the epididymal fat is in the inguinal region! 𝑥̅ =6,44×106±7,44×106 from mouse and x108 from rat!
The MSCs from fat always have a good yield!
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I am generating u87 Gliblastoma stem cells and I want to see their differentiation ability.
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I want to differentiate MSC in the tri lineage tissues in an in vitro system. May I use human insuline to differentiate those cells in adipogenic tissue?
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Dear Helena 
You should be differentiated by ibuprofen and beta glycerol phosphate composition. 
after this phase, you should be used oil red staining for the final check.
Good luck 
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I would like to isolate mesenchymal stem cells from mouse bone marrow but I am confused with all different protocols I found in papers. Ηas anyone any idea about a simple protocol for isolate and culture of this cells?
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cut the ends of the leg bones and flush the marrow out using insulin syringe. Culture in stemcelltechnologies "mesencult proliferation kit" according to their protocol. If you don't want to buy their expensive medium you need someone who knows what FBS lot number to buy, MSCs won't grow in most lots. Other than that, be very patient, it can take ages (<3 months) for the cells to start growing well (mouse MSCs undergo a transformation at ambient oxygen levels, the only way to avoid that is to have access to an incubator that can reduce oxygen to 5%).
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I want to ask a basic question in stem cell research.How scientists isolate and identify new type of cell population from  total cell population in culture dish as nothing is known about it  like marker etc, for isolating that cell type.
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thats the problem..no marker is known.
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I'm wondering if someone used or is going to use nanoflares (smartflares) to isolate cells based on target mRNA sequences.
I'm trying to do it but still have some problems.
Could anyone help me?
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We have now published our article evaluating the SmartFlare technology
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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.
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I only cultured the stem cell from mouse SVZ, but I think the procedure will be similar.I advice you see the video on JOVE "isolation and culture of mouse neural precursors ", Hope it will help
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In BALB/c mice treatment of Ethylnitrosourea can produce leukemia but both lymphoblast and myeloblast cells are present. How Can I isolate only lymphoblast or only myeloblast cells from the bone marrow or from the peripheral blood?
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For human leukemia: NO - aberration means no specificity; for ENU induced leukemia: you have to find out.
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I want to isolate the cancer stem cells from cancer cell lines. But most of the people are isolating the cancer stem cells by using FACS. But in my lab it is not available. So I want to use another efficient methods to isolate cancer stem cells. I am hopefully expecting effective answers and suitable protocols for that.
Thanking you all. 
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One way researchers enrich for cancer stem cells is by culturing in non-adhereant, sphere forming conditions. By doing so you do not get a pure "stem cell" population but you do enrich for "stem cells" which can be passaged multiple times. Here is a publication on the idea using primary breast tumors.
Hope this helps.
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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?
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Dear Experts I am trying to isolate stem cells from an inflammed oral tissue. In usual practice we use 0.5% -1% of antibiotic ( Penstrap) for healthy tissue. In order to isolate cells from inflammed tissue we increased the concentration of antibiotic to 2% in washing buffer as well as in culture media. However, we are not able to control the contamination/ infection in the flask with inflammed tissue. Please suggest the way to address  the issue. 
Thanks 
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I used to wash tissue before sampling with a iodine disinfectant then washing the sample in a 2X AB solutions (I used DMEM for buffering). Then you should be ok.
Good luck
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Hello,
I am new to the stem cell biology field and I need to set up a cardiac differentiation experiment using mES cells. I have looked at various sources and have some ideas about what mES cells to use, but I thought I would ask if anyone had personal experience with a mES cell line they loved and would recommend to start with. I am on a budget, and I will be the only person in my lab working with mES cells. 
Thank you,
Best wishes, 
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Hi Adam, I'd like to share these two protocols with you and I bet they will help you a lot.
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hello,
anyone knows a protocol for culturing (salivary gland) stem cells on a transwell membrane , culture inserts?
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You are welcome
 Best Regard
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I'm trying to isolate human HSC from cord blood. I use Ficoll to get  mononuclear cells and then perform lineage depletion,CD34+ seletion at last. Yet I found that early stage erythroblast as nucleated cells couldn't removed by Ficoll density centrifugation. Also they couldn't be remove by lineage depletion. For the worst, they express some extent level of CD34. So I get huge contamination of early stage erythroblast in CD34+ HSC. I know that Rosette form STEMCELL Technologies could remove those cells before CD34+ selection, besides that,any idea on how to remove these cells, like unique markers?
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I suppose it is fine to use other kind of red lysis since it is a regular and simple reagent with NH4Cl. I did use a red lysis buffer from another vendor and it didn't work well for unknown reason so we keep using PharmLyse and never got a problem. Using Dynabeads is a popular magnetic isolation for years. It should be fine. My colleague here used this method and it works fine (not for HSC). However I always use Miltenyi system since I learned it from my old lab.
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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!
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The thawing procedure are as following:
1. Warm the medium for 20 minutes in a 37° C water bath.
2. Place 5 ml of the warmed fresh medium into a new 15ml centrifuge tube.
3. Thaw the cells in 37° C water bath by gently spin around the vial within 2-3min. During this process, the cap is not immersed into water. 
4. Carefully extract the cells with a pipette and transfer them into15ml centrifuge tube which is placed 5ml medium in advance. And then mix them fully.
5. Centrifuge: 1500rpm 3min 4° C. 
6. Dessert the supernatant and re-suspend the cells with 10ml medium and then transfer them into 100 mm dish.
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Thanks Fey !!  I would implement your suggestion in immediate terms and get back to you with the results !!
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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?
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Neurobasal media may not be the best choice for e-phys. You also do not mention if you add BDNF or GDNF to your final differentiation medium...  You could maybe try one f the commercial available e-phys media, but if your neurons are not healthy, this needs to be addressed first..
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Please help with me a working protocol for extraction of stem cells from adipose tissue?
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 If you are working for a stem cell bank you probably don't want to use an enzymatic digestion. Perhaps this publication can help: http://www.ncbi.nlm.nih.gov/pubmed/23725689
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I have been trying to isolate neural stem cells from periventricular tissue (taken from an adult patient). But I do not get any attached cells after 2-3 weeks of culture in NB medium supplemented with glutamine, B27, bFGF, EGF and heparin on PDL/Laminin coated flask. The protocol was carried out as described in a paper on JOVE with slight modification.
1) During tissue dissociation, the tissue (of 10 pieces 1mm size from endoscopic needle sampling) was minced briefly and digested in 1 mL 0.05% Trypsin EDTA with DNase I at 37C for 5 min.(No trituration at this step)
2) The tube was swirl several time during incubation followed by addition of trypsin inhibitor.
3) The pellet was spun down at 1500 rpm, 5 min and supernatant was discarded.
4) The cell pellet was resuspended with growth medium (as mentioned above) and filter through 40 um cell strainer.
5) The cell suspension was spun down again and the pellet was resuspended in growth medium.
6) The cells were seeded at density of 250000 cells in a PDL/Laminin coated T25 flask.
However, we only observed formation on cell clumps after several days of culture and those clumps were not neurosphere.
Is there any step that I need to improve or revise for better result?
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I think my be you need to extract NSCs from tissue using papain dissociation system, which works good for us. After couple weeks culture in growth media you should get pure neural stem cells. they should be attach after then.
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Many methods are already published to isolate urine derived stem cell, any suggestion for the best method to isolate urine derived stem cell? is there any commercial kit pack to get it? 
best regards
Yudi
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From what I gather, people collect urine (200 ml or so) and pellet the cells and debris in a low speed centrifuge then just plate out the cells in media.  For specifics on the types of media see:
Guan et al.  PLoS One. 2015 May 13;10(5):e0125253.
what these cells really are or where they came from is not entirely clear to me. 
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Im currently working on histology and anatomy of dental apparatus of different species of cartilaginous fish and looking for colleagues to share thoughts and ideas.
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Thanks a lot, looking forward to receive your paper. God bless
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what surface markers are used to differetiate between a normal stem cell and CSCs and CTCs?
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Circulating tumor cells (CTCs) have the ability to form the tumor tissue in the pre-metastatic niche, which is why CTCs are believed to be similar to cancer stem cells (CSCs). CSCs are defined as to harbor self-renewal ability and multi-lineage differentiation potential. CSCs tend to be de used to explain the formation of intra-tumoral heterogeneity in the perspective of genetics/ epigenetics as well as susceptibility to anti-tumor therapies. On the other hand, CTCs tend to be used to explain the multi-step metastatic cascade composed of dissociation from the primary site, intravasation, circulation, extravasation, and colonization/ proliferation at the distant metastatic foci.
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I am trying to standardize a protocol of stem cells from dental pulp from exfoliated deciduous teeth, but would like an protocol that not use animal serum. any of you have any serum-free protocol or with human serum ?
 and the suggestion would be autologous human serum ?
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Hello Bruna,
To be very brief and specific. You can use human serum in the range of 5-10% but human serum is reported to increase the osteogenic potential of dental pulp stem cells. So if that is not an issue, you can successfully opt for human serum. But if you want to grow them without serum supplementation you have to supplement the culture media with other supplements like EGF, PDGF etc. Best option to avoid animal serum and to fullfill the nutrient requirements of cells is use of Knockout serum replacement (KSR). And apart from the typical collaginase/dispase digestion I will suggest you to go for explant culture as a more pure population can be obtained from the same.
Hope it helps !
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I have Neural stem cells (derived from adult mouse HC) in a neurosphere culture. As I saw changes in proliferation in my knockout in vivo after depolarisation, I would like to repeat this experiment in vitro. 
I would like to depolarize the cells with KCl and afterwards do a BrdU-proliferation-assay. I want to see the changes in the type1 cells - not in differentiated neurons. I would first plate the dissociated neurosheres on cover slides coated with matrigel (in medium) over night to let them settle down and then change the medium with medium containing KCl.
But I find very different protocols concerning concentration of KCl (between 15 and 100um) and duration (6h to 6days). 
Do you have any suggestions on wich concentration and duration I should use?
Thanks!
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Zhang Yan is correct in that concentrations and durations can vary widely, and that for chronic administration, a lower concentration is typically better. I have only used high KCl to depolarize primary neuronal cultures, so the optimal concentrations may be different for your neurospheres. I used 50mM KCl for short-term treatments (up to 1 hour). I have never chronically depolarized my cultures (chronic depolarization changes vulnerability to excitotoxicity, a change which I was trying to minimize), but if I were going to chronically depolarize neurons,  I would probably start with 10mM KCl.
If I were you, I'd first decide on a duration of treatment and then do a dose-response curve for that duration to see which doses cause the level of depolarization that you want to see. So the question becomes, is it better for your experiment to have a short period of intense depolarization, or a sustained but more moderate depolarization? 
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Followed by the protocol of Science paper “Pluripotent Stem Cells Induced from Mouse Somatic Cells by Small-Molecule Compounds”,the D18 morphology of inducing MEF cells as pictures show,it seems like the retrovirus-induced MEF on D5. Last time I followed the protocol to continue in the chemical-induced medium and 2i-medium on D40,but failed to generate cips.I think maybe, like the retrovirus-induced methods,these clones could be replated on feeders now,but considering lack of exogenous factors stimulation, this may not be appropriate.
Can anyone give me some suggestions about this? To repeat the protocol or do some changes.thx. 
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Don't understand your question.  Did you get Deng HK ciPSC protocol to working using his 7 small molcules as described in his Science paper “Pluripotent Stem Cells Induced from Mouse Somatic Cells by Small-Molecule Compounds ?
I don't think anyone has so far managed to do it.  And I am in that camp!
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I am trying to isolate umbilical warthon's jelly MSCs and I find some populations that forms embryoid body like clusters. I have never seen this ones before when working with AD-MSC. I am wondering if it is a different population.Thanks
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Hi Ramon,
MSCs tend to form that sort of colonies (or clumps), it' s a feature of fibroblastoid cells (mesangial cells and fibroblast tend to due the same, even tough at a less rate), these colonies are not embryoid bodies and they are not pluripotent at all, if you analyze them (FACS or IF), you will see that they are exactly as the other MSCs that grow as a single layer, there is no difference at all.
Let me know if you need anything else,
Letizia
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Shall I change to any other harsh lysis buffer? Also I have run the gel on SDS PAGE  I am getting one single band between 50- 75 Kda. Is it because of improper denaturation? Please help.
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Can I ask you from which kind of flask/plate you start and the lysis volume you incubate the cells in?
In our experience, even if we use a NP-40 based buffer, a volume of 70-150 ul works well with 25cm2/75cm2 flasks. In this way you should obtain protein concentrations ranging around 10 ug/5ul, so easy to load 30ug for a good western.
Please consider that MSCs are larger than standard cultured cells (e.g. Hela) so even a confluent flask has a lower yield (in numbers) than expected.
Protease inhibitors may be in a cocktail (many are readily available as 100x solutions from different providers), and not limited to PMSF alone, as tipically MSCs and culture media bear also metaloproteinases and other proteolytic activities which are insensitive to PMSF.
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Currently I am working on isolation of mesenchymal stem cells from human umbilical cord. Since MSCs have the unique properties of self renewal and differentiation into various lineages, my protocol demands to keep them undifferentiated. Nanog and LIF have been mentioned as factors responsible for undifferentitation of MSCs. However their roles are not clear. Kindly help.
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Hello,
I worked on isolation and culture of WJ-MSCs and our main culture protocol is mainly adherent to those published by others. Low-glucose DMEM, plus 10% FBS, NEAAs, antibiotics, and cells can be maintained up to passage 15th, maintaining morphology and markers expression.
In these conditions, if you maintain the standard passaging ratio, the medium alone will not be sufficient to promote any differentiation process. Never tried one of the mentioned factors: in our hands cells do not need them.
Hope to have been of help.
Giampiero
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How can I check whether I can use that or not?
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Hello,
I use routinely human VEGF for my mESC differentiation protocol towards blood and endothelial cells. I use the VEGF from R&D systems (#293-VE-010). I think it should work well on your mouse primary cells.
Good luck.
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Hi all,
I'm working on differentiating ESCs to a forebrain lineage and was wondering how to calculate differentiation efficiencies?  I see efficiencies written in some ESC induction papers (example, some papers quote a 20% differentiation efficiency from their ESCs) but am not sure how these are calculated.
Are they from calculating the number of cells in the final plate (at the end of the differentiation time line) that are positive for a lineage marker vs. the number that aren't?
Example: Final plate is stained with an antibody against a lineage specific protein.  20% of the cells in the field of view (based on DAPI) is positive for this marker.  So efficiency is 20%?
Or is it based on number of starting cells divided by number of cells in the final culture positive for a lineage specific marker?
Example: Start with 10000 ESCs dissociated and plated.  Count number of cells throughout differentiation protocol (assuming that cells are dividing at least initially after plating out, lets say cell number plateaus at 1 million).  After differentiation protocol, end with 10000 cells that are staining positive for the lineage marker.
Would the efficiency then be 10000 (# marker positive cells) /1000000 (cell number at plateau) = 0.01, or 1%?
Or is there some other (probably smarter) way of calculating differentiation efficiency?
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Hello Amy,
You can't count  around 10,000 cells positive for a specific marker  under a microscope and also you are going to compromise your accuracy by same. To be very specific and brief, you can calculate the percentage positivity for a marker which is positive for that partiular lineage by FACS.. Percentage positivity gives you a direct indication of differentiation efficieny. Suppose at control level ESCs are expressing MAP-2 which is a mature neuronal marker, as 0.5% and after differentiation protocol its 95.5% then obviously the efficiency of ESCs to differentiate into mature neuron is 95%. Through FACS you can get a direct record of postive percentage and no need to go through complicated formulas or equations...
Last but not the least, comparision of control and differentiated population is must to get final accurate value of percentage efficiency and your marker of choice should be highly specific for that lineage.
Hope it helps..!
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Is the use of CD51 , CD31 and Sca-1 in combination with depletion of CD45 and other lineage markers ok?
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Salam alaikum
For isolating MSC from mouse bone marrow follow this protocol :
1-Euthanize mice by CO2 asphyxiation. It is preferable to work with 2-3 mice at
one time. To generate MSC cultures, utilize pools of 4-5 mice, of which arebetween 6-10 weeks of age.
2. Harvest hind limbs from one mouse. Remove skin and as much muscle and
connective tissue as possible. Cut the limb above the hip and below the ankle
joint (including some of the foot); it is important to maintain the bone ends to
ensure sterility of the bone marrow. After severing theh limb, carefully break
apart the knee joint and strip remaining connective tissure from both ends of
femur and tibia if ends remain intact.
3. Briefly wipe bones with gloved fingers dipped in 7-% EtOH and place bones in dish of sterile 1X PBS on ice. Collect all bones in pool in same dish.
4. Move pooled dish of bones into sterile tissue culture hood. Wash bones by
transferring through sterile PBS 6 times.
5. Snip off ends of each bone with scissors (keeping as close to end as possible to extract more bone marrow) and gently place in sterile PBS.
6. Fill 10cc syringe with prewarmed Complete conditioned media and attach 25
gauge needle. With forceps, grab one bone. Use syringe to force media through
bone shaft to extract all red marrow into 150mm plate. Can flip bone over and do from opposite side. Repeat a few times to ensure all marrow is removed. Boneshould look very white when finished. Continue until all bones are demarrowed.
7. Pipet cell mixture up and down a few time so dissociate cells- can also use
syringe to pull large marrow pieces through needle to dissociate further.
8. Pass cell suspension through cell strainer (70µm size) to remove any large cell clumps or bone particles.Count cell suspension. Add 10µm cells to 10µl trypan blue. Use hemacytometer to count.
plating for antibody staining or other experiment directly, plate at 1x106
per well of 6-well dish.
• If maintaining for future experiments, bring total volume to 25-30mL and
plate entire volume in 150mm plate.
9. Incubate cells until cells adhere and look nearly confluent—usually around day 4 of culture for a pool from 4 mice. Remove media and wash once with PBS to remove nonadherent cells. Trypsinize and split 1:3- 1:5.
10. Expand cells until 70%-90% confluent, changing media every 3-4 days. At this point, can either split further for experiments of freeze in liquid nitrogen for further use.
11. At this time, cells can be plated for different assays including differentiation
antibpdy staining on gelatinized coverslips, and tolerance to cellular stresses
such as ionizing radiation or serum starvation, for example.
NOT : Of The Solutions:
Complete conditioned media with 10 % FBS- 500mL
500 mL a-MEM media (Invitrogen)
50mL FBS (it is important to stick with a serum lot that works well)
5mL Pen/Strep solution (100X)
5mL L-glutamine (100X)
Filter and store as usual.
please , Read the attachment to see all CD marker for the mouse, also IF you need the specify the method used to detect these cells ( IHC , Elisa , Western Blot ,flow cytometry ...et ) So that we can help you in protocol for cell staining .
Good Luck 
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I have tried mechanical  and chemical protocols on adipocytes but still the yield is scanty and they are slowly growing. I can't get enough population to grow. I used platelets rich plasma and FCSs alone in the first 12 hours but it's not working.
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Hello Amira, 
I suppose you are talking about adipose tissue right ? I've tried, mechanical, enzymatic and explant cultures to extract hMSCs from adipose tissue and the less time-consuming and the most cost effective way is the explants method. I can provide more information if you want but the protocol is fairly well explained in N. Priya et al., 2012, J Tissue Eng Regen Med. I applied this method to human, canine and mouse adipose tissue. I performed extended immunophenotype characterization (+30 markers) at the end of P1 on the hMSC and the cells fit the recommended ISCT MSC phenotype, they can also differentiate well into osteoblasts, adipocytes and fibrocartilage. In serum-free conditions I maintain a doubling time of approx. 35h during the 6 first passages and approx. 40h with bFGF-supplemented, FBS-containing medium. I haven't tried culturing them in platelet lysate yet.
Hope it helps don't hesitate to get back at me. 
hF
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I currently want to dissect a rat and store its organs inside liquid nitrogen for future cells or stem cell extraction, including heart, liver, kidney, bone marrow, tendon, cardiomuscle, etc. I have questions:
1) What kind of media should I use?
2) Can I store them in -86 C Freezer instead of LN2 for long periods of time (few months)?
3) What is the precaution I should notice during the thawing process?
Thanks a lot for your answers,
Long.
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Hey Long,  look at the two links below that describe the best ways to preserve your tissues for cell culture down the road.  Hope this helps.
Exp Toxicol Pathol. 1999 May;51(3):229-34.
Cell cultures from cryopreserved renal biopsies and other tissue samples.
Sommer M1, Fünfstück R, Stein G.
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I tried to obtain bone marrow-derived mesenchymal stem cells of mice. However stem cells don't adhere to the culture dish after the first passage. Maybe anybody faced this problem.
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BTW Matrigel and not materiel in my previous comment. My phone mistakingly corrected it.....
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When i have checked the proliferation marker (Cyclin D1) in cancer stem cell (CSC) then its expression was higher. However when i have developed the in vivo tumor which is highly aggressive and re-checked the status of cyclin D1 in these total tumors lysate by western. Result showed that there were no change of cyclin D1 expression in CSC in contrast to non-CSC, irrespective of tumors volume.
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The in vitro growth is higher because there are no interactions with tumor stroma, normal cells etc.
In vivo conditions are more complex e.g, the presence of competitor cell populations or quorum sensing-like signals  modify the growth conditions not only for cancer stem cells but the activity of many other cells will change.
 .
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I am doing the mesenchymal stem cell isolation from rats. I wonder whether there are differences between cells extracted from neonatal rats and from those from older rats. First of all, I found that older rats have bigger cells than the younger one by looking under microscope. Do you think it is true in general? And are there any other differences between neonatal MSC and "old MSCs"? Thanks a lot for your sharing.
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Hello
There are many studies that provided evidence of that there are age-related changes in MSCs derived from the bone marrow of old rodents with regard to plasticity and basic stem cell biology.
see the article in attachment
Good Luck 
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We are planning to evaluate CD34+ cells from a cell line but isolation requires enormous amounts of cell culture of the AML cell li