Science method
Stem Cell Isolation - Science method
Explore the latest questions and answers in Stem Cell Isolation, and find Stem Cell Isolation experts.
Questions related to Stem Cell Isolation
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.
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.
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?
Pericyte and SMC have same marker and functions. I can't find any publications to differentiate pericyte from SMC?
Hi I would like to isolate CD34- (negative) cells from cord blood and culture them? Can anybody share their experience with isolation and culturing?
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.
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?
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.
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.
This is the positive fraction. I don't know if it's contamination or just debris of dead cells

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
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
I read some papers that use G-rex or conventional tissue culture flasks. Which one do you suggest? Which medium is the best?
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.
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
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!
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.
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.
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.
What collagenase enzyme should be used to isolate human adipose derived stem cells and what concentration should be use on liposuction material?
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
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?
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!
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.


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
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.
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.
What precautions one should take to maintain viability of stem cells of adipose tissue, during their isolation
Is there a good kit to isolate stem cells from cancer cell line
myeloma stem cell
isolation
sorting
culture
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?
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?
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?
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
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
I am generating u87 Gliblastoma stem cells and I want to see their differentiation ability.
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?
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?
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.
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?
How can we detect CML Stem Cells, and how can we isolate from CML patients?
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.
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?
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.
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?
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

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,
hello,
anyone knows a protocol for culturing (salivary gland) stem cells on a transwell membrane , culture inserts?
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?
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!
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?
Please help with me a working protocol for extraction of stem cells from adipose tissue?
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?
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
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.
what surface markers are used to differetiate between a normal stem cell and CSCs and CTCs?
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 ?
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!
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.


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
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.
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.
How can I check whether I can use that or not?
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?
Is the use of CD51 , CD31 and Sca-1 in combination with depletion of CD45 and other lineage markers ok?
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.
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.
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.
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.
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.
We are planning to evaluate CD34+ cells from a cell line but isolation requires enormous amounts of cell culture of the AML cell li