- Saleh Alkarim added an answer:Can anyone suggest a non-invasive procedure to obtain fibroblasts from patients?
We will need primary cells from patients that have a rare inherited metabolic disease. We will be turning them in to IPS and then differenciate them into hepatocytes. We have to use a non-invasive method to obtain primary cells and right now we are planning isolate keratinocytes from hair bulbs following Aasen & Belmonte's procedure ( http://www.nature.com/nprot/journal/v5/n2/full/nprot.2009.241.html ) but since working with keratinocytes is somewhat more tedious and expensive than working with fibroblasts, we would like to be able to isolate fibroblasts via a non-invasive method.
Can anyone suggest such method or alternative?
Also would it be possible to obtain fibroblast culture from hair bulbs?
agreed with Pavel Makarevich
At least there is less risk of cutaneous .
Please read the article in attachment , may help you .
- Joe Graymer added an answer:Why do cancer stem cells sometimes exhibit high proliferation in vitro but exhibit less proliferation when placed in vivo in a tumor condition?
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.
In these cases, and I don't have any Lab experience in Cancer Cells, I'd say that seed, the cancer cell, is the same, but the soil, the Petri dish or a living thing tissue, differs.
From immune interactions to the microenvironment changes induced by the tumor cells, that have different actions when the cells are isolated in a culture or inside a tissue, many issues could be addressed as research subject in your question, that may have many types of answers.Following
- Inese Cakstina added an answer:Stemness marker for human bone marrow derived MSC - can anyone help?Currently so many people are working with mesenchymal stem cells and they use surface markers for the validation of cells to be positive for CD90, CD73, and negative for CD45 and other kind of markers. But with none of these markers you can be really sure that you have real MSC WITH STEMNESS QUALIFICATIONS. There are so many articles and people use a huge set of markers to prove they have MSC and it is also very difficult to dig up the right information because of a huge number of articles. I want to know based of your experiences can you suggest a marker which is easy to go? And in other words is there even any marker? I also know about Stro-1 and CD 105 but these Markers are not real marker for stemness.
I found this article interesting - sorting MSC's by physical parameters: http://www.ncbi.nlm.nih.gov/pubmed/25411477
Here is the press release from MIT: newsoffice.mit.edu/2014/identify-rare-stem-cells-in-bone-marrow-1006?elq=1540593920034fc997f3c32c69bf7875&elqCampaignId=4Following
- Maurice Canham added an answer:Have you seen a difference between E8/Vitronectin and mTESR/Matrigel for iPSC, in terms of differentiation ability or chromosomal stability?
We have observed several iPSC clones with chromosomal abnormalities grown in E8/Vitronectin and poor neuronal differentiation ability and wonder if this is purely co-incidence or a culture effect.
We have noted that the high cntent of FGF2 present in Essential 8 can indeed be inhibitory to differentiation. Seeding cells in a low fgf containing media (like Nutristem) can overcome this. Alternatively, we have tried seeding in E8 but then adding PD03 for one day and this also helped our protocol. I'm not sure about the effects of vitronectin versus matrigel since we are using Laminin 521. In terms of chromosomal abnormalities, do you directly compare karyotpyes (or SNP data) of cultures of E8/Vitronectin and mTESR/Matrigel at the same passage and find a difference? Abnormalities can increase with passage for hEPCs. See attached papers relating to fgf signalling and self-renewal/differentiationFollowing
- Marin Jukic added an answer:Can someone point on a good review/articles describing signaling cascades in colon development in mouse embryo?
Wnt, Hh, Bmp's etc. Gene-expression/ protein changes marking stages in normal colon development
Thanks a lotFollowing
- Islam Saadeldin added an answer:What are the factors affecting stem cells differentiation in vivo or in vitro?There are several factors affecting stem cells differentiation during embryonic development stage. I would like to discuss these factors together through your research and your experiences. Either in vivo or in vitro .
From my experience; using feeder cells maintain the normal morphology of oligopotent ESCs (trophoblasts) however using Matrigel (basement membrane matrix) stimulate differentiation into elongated spindle like cells.
I would like to share a time-lapse video I captured during my experiment.
- Vahid Razban added an answer:Why are mesenchymal stem cells obtained from mouse adipose tissue spread in morphology?
Why are mesenchymal stem cells obtained from mouse adipose tissue spread in morphology?
I am culturing Mesenchymal stem cells from mouse adipose tissue (epididymal fat tissue).Fat tissue is minced properly and digested using 0.05% collagenase type 4. The cells are then seeded in T-25 flask in IMDM 10% FBS containing media. Culture media is then changed after 48hrs. Cells trypsinized when 80% confluent using 0.25% trypsin.
Phenotypic surface marker expression of MSC at P-3 , CD29= 78.1% ,CD73= 21.2% ,CD44= 6.78% ,SCA1= 21.8% ,CD31= 4.22%.
Is it that MSCs obtained from adipose tissue are spreaded in morphology?
Do I need to change the culture conditions?
Murine mesenchymal stem cell isolation differs from human counterparts isolation at least in some parts. Murine MSCs seems to be immediately enter a sleep period during which cells start to die, detach and decrease in number. After several medium replacements (2-3 times/week) they start to proliferate and gain to their spindle shape. Its my experiencesHope to be useful.Following
- Toks Yerokun added an answer:Can 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
I will also check to make sure that what is being called stable is really stable, which means the vector is incorporated into the genome.Following
- Yves Gérard Illouz added an answer:Do all patients' adipose SVF cells freeze?
We have noticed that some patients' cells freeze and thaw easily and those patients get good clinical response to frozen cells. There is a group who do not respond well to frozen cells. In the vet world we see some bulls whose semen will not freeze. Is this the same for human SVF cells?
Normally, no problems !Following
- Andrew S Brack added an answer:Is there evidence for non-satellite cell resident muscle progenitors?On ResearchGate, there has been a very collegial discussion about the role of satellite cells during muscle hypertrophy. A number of us also cited increasing evidence that non-satellite cells play a role as niche support cells and/or can directly participate in the myogenic lineage. So, hoping for a fruitful discussion, lets start to put this data together to see if it forms a coherent picture? Jump in.
Thanks for clarifying.Following
- Henry E Young added an answer:What is appropriate media for MSCs culture?
I would like to know using Alpha MEM having ascorbic acid is good idea or not for mesenchymal stem cells culture. I generally use mice/rat bone marrow stromal cells for MSCs. using alpha MEM containing ascorbic acid induces differentiation of MSCs. I would be happy to know which media is best for MSCs culture.
I culture with Opti-MEM with Glutamax (GIBCO) with (1-10%) heat inactivated serum (Atlas Biologicals, Fort Collins, CO), pH 7.4 (+/- Pen/Strep) (base medium). Works fine and keeps cells from differentiating. If you want cells to proliferate add PDGF-BB (R&D Systems), if you want induction add appropriate recombinant inductive factors, i.e., BMP-2, VEGF, FGF-alpha, TGF-beta, basic-FGF, etc. to base medium.Following
- Sandeep Rajput added an answer:Which marker is best for staining of breast cancer stem cells by immunohistochemistry?
I have been collecting breast tumor tissues from patients and am going to study the expression profiling of stem cell regulatory genes. Also, i would like to do assessment of breast cancer stem cells in tissue section by immunohistochemistry.
but am in little bit of confusion regarding markers. Which marker is best for assessment ? ( ALDH, CD133, CD24, CD44)
You can also include ESA (Epithelial specific antigen)Following
- Elena M Glinka added an answer:What kind of plant nanofibers have positive and negative effects on human stem cells?I want to now which plants nanofiber has not genotoxicity effect on human cells
According to de Lima et al., (Int J Nanomedicine. 2012;7:3555-65) and (Clift et al. (Biomacromolecules. 2011 Oct 10;12(10):3666-73) cellulose nanofibers isolated from cotton did not cause any significant DNA breaks in the cell types employed (lymphocytes, fibroblasts, human lung cells).
- Sayandip Mukherjee added an answer:Can someone answer the questions below regarding correct culturing procedure for the reprogramming of mouse iPSC ?
Additional details include multiple questions:
1) Can mouse iPSC be cultured in feeder free conditions. Kindly provide the details of the media and matrix that could be used for the feeder free culture of mouse iPSC.
2) Passaging of mouse iPSC can be done using Trysin?? Or other methods including mechanical dissociation/milder enzymes such as collagenase or dispase are required??
3) Can the somatic cells after protein transduction be directly maintained in feeder free condition with the media supplement of the conditioned iPSC media from mouse MEF's?? Or its is important to grow cells in presence of feeder layer ducring initial reprogramming phase???
You will get all your answers in this publication:
Hope that helps!
- Steingrimur Stefansson added an answer:What buffers are safe for in vivo use?I read a study (link below) where they cultured cells and re-suspended them in a buffer before injection. I am curious what buffers are safe for this? PBS? aMEM? What do you think?
Hi Ellie, if you are going to inject cells into animals, the best way is to use tissue culture grade media, DMEM, EMEM, whatever.
These are sterile media that have minimal endotoxin and other pyrogens in them.
You just have to be careful about using sterile techniques to prep. the cells for injection.Following
- Constantine Kaniklidis added an answer:Anybody knows a good review on HP-1 and its role in cell senescence?
I am interested in the role of HP-1 in cellular senescence. We know that HP-1 is involved in the formation of senescence associated heterochromatin foci (SAHF), although it does not bind DNA domain. Nothing is known about its regulation and cellular localization in different cell types. I was wondering if someone is aware of a a good literature review on HP-1 and cellular senescence.
There are a number of important papers1-19 in this fascinating arena - in addition to more general reviews12-21 - so I will briefly outline below some of the more relevant literature, and references within these will allow you to pursue this vital topic further.
Cells from aged humans experience a decline in the levels of the heterochromatin 1 proteins (HP1) which are a component of senescence-associated heterochromatin foci (SAHF)1,2. In this connection, important are the results of the recent University of Rochester genetic study of heterochromatin formation in Drosophila3 that strongly demonstrate that heterochromatin levels - with HP1 being an integral component of heterochromatin and in fact controlling heterochromatin levels4,5, which in turn control rRNA synthesis [Larson, above] - significantly influence and prolong life span, with moderately higher levels of heterochromatin promoting longevity, given that it is confirmed that heterochromatin levels decrease with normal aging, and that heterochromatin formation is essential for silencing rRNA transcription (this transcription promoting protein synthesis in general) via repression of unnecessary rRNA synthesis in order to promote genome stability, and longevity.
And investigators at the Fox Chase Cancer Center have demonstrated that HP1 proteins, prior to incorporation into SAHF, are transiently localized to PML bodies, where they colocalize with the chromatin regulator/histone chaperone HIRA6. More importantly, recruitment of HIRA into PML bodies is one of the earliest steps towards senescence in human cells , and we know that blocking entry of HIRA to promyelocytic (PML) bodies prevents formation of SAHF7, and HP-1 is centrally involved in the formation of ALT-associated PML nuclear bodies (APBs)8 playing a role in telomere-associated senescence.
The Cancer Connection:
Along a parallel line of inquiry, many investigators have also pursued the implications of HP1 and SAHF in the oncology context, leading to the hypothesis that the role of SAHF is to counter DNA-damage response (DDR) in order to force the cell into senescence (as opposed to apoptosis) and suggests that SAHF-associated heterochromatinization might be a barrier to tumorigenesis, as suggested by the fact that several cancer cell lines display elevated HP1 levels9. Therefore, opposing senescence-associated changes in heterochromatin HP1 (and H3K9) occur, consonant with the fact that age is a significant risk factor for cancer10.
Narrowing the Role of SAHF/HP1:
However, against all these views, it is critical to note that recent results11 have marshaled evidence that, unlike the DNA damage response marker γH2AX, SAHF is itself not a common feature of cellular senescence - indeed, that SAHF formation is wholly dispensable, and NOT, as commonly first thought, a universal component of senescence, for multiple forms of cellular senescence - and despite the fact that SAHF formation is shared by diverse cultured cell types under oncogenic stress, it nonetheless appears that SAHF are cell-type-restricted under genotoxin-induced and replicative senescence, and that in consequence, again against widely assumed views, that SAFH does NOT represent a reliable common marker to identify cells undergoing senescence in general (it would appear that only γH2AX-positivity, marking constitutive DNA damage signaling, is a reliable senescence marker), leaving a more narrow purpose for SAHF formation in oncogene-induced senescence.
Where We Stand, and Going Forward
Thus, we have evolved to a more nuanced understanding of the more limited role of SAHF and heterochromatin 1 proteins (HP1) in cellular senescence, although it nonetheless remains true that the heterochromatin process is vital, through elevated heterochromatin levels, in promoting genome stability, and longevity via silencing rRNA transcription and thus the repression of unnecessary rRNA synthesis, and it is clear that we will surely have other studies pursuing the exciting field of how heterochromatinization can be modulated to induced prolongation of lifespan, and - in parallel - suppression of tumorigenesis, thus advancing positivity both against aging and cancer.
- Scaffidi P, Misteli T. Lamin A-dependent nuclear defects in human aging. Science. 2006;312:1059–63.
- Cao K, Capell BC, Erdos MR, Djabali K, Collins FS. A lamin A protein isoform overexpressed in Hutchinson-Gilford progeria syndrome interferes with mitosis in progeria and normal cells. Proc Natl Acad Sci USA. 2007;104:4949–4954.
- Larson K, Yan SJ, Tsurumi A, et al. Heterochromatin formation promotes longevity and represses ribosomal RNA synthesis. PLoS Genet 2012; 8(1):e1002473.
- Grewal SI, Elgin SC (2002) Heterochromatin: new possibilities for the inheritance of structure. Curr Opin Genet Dev 12: 178–187.
- Ebert A, Schotta G, Lein S, Kubicek S, Krauss V, et al. (2004) Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev 18: 2973–2983.
- Zhang R, Poustovoitov MV, Ye X, Santos HA, Chen W, et al. (2005) Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA. Dev Cell 8: 19–30.
- Ye X, Zerlanko B, Zhang R, Somaiah N, Lipinski M, et al. (2007) Definition of pRB- and p53-dependent and independent steps in HIRA/ASF1a-mediated formation of senecence-associated heterochromatin foci. Mol Cell Biol 27: 2452–2465.
- Jiang WQ, Nguyen A, Cao Y, Chang AC, Reddel RR. HP1-mediated formation of alternative lengthening of telomeres-associated PML bodies requires HIRA but not ASF1a. PLoS One 2011; 6(2):e17036.
- Di Micco R, et al. Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer. Nat Cell Biol. 2011;13:292–U244.
- O'Sullivan RJ, Karlseder J. The great unravelling: chromatin as a modulator of the aging process. Trends Biochem Sci 2012; 37(11):466-76.
- Kosar M1, Bartkova J, Hubackova S, Hodny Z, Lukas J, Bartek J. Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16(ink4a). Cell Cycle. 2011 Feb 1;10(3):457-68. Epub 2011 Feb 1.
- Burgess RC, Misteli T, Oberdoerffer P. DNA damage, chromatin, and transcription: the trinity of aging. Curr Opin Cell Biol 2012; 24(6):724-30.
- Krishnan V, Liu B, Zhou Z. 'Relax and Repair' to restrain aging. Aging (Albany NY) 2011; 3(10):943-54.
- Lazarus A, Banerjee KK, Kolthur-Seetharam U. Small changes, big effects: chromatin goes aging. Subcell Biochem 2013; 61:151-76.
- Feser J, Tyler J. Chromatin structure as a mediator of aging. FEBS Lett 2011 Jul 7; 585(13):2041-8.
- O'Sullivan RJ, Karlseder J. The great unravelling: chromatin as a modulator of the aging process. Trends Biochem Sci 2012; 37(11):466-76.
- Chung I, Osterwald S, Deeg KI, Rippe K. PML body meets telomere: the beginning of an ALTernate ending? Nucleus 2012 May-Jun; 3(3):263-75.
- Corpet A, Stucki M. Chromatin maintenance and dynamics in senescence: a spotlight on SAHF formation and the epigenome of senescent cells. [Chromosoma 2014 May 27.
- Klement K, Goodarzi AA. DNA double strand break responses and chromatin alterations within the aging cell. Exp Cell Res 2014 Sep 8.
- Tsurumi A, Li WX. Global heterochromatin loss: a unifying theory of aging? Epigenetics 2012; 7(7):680-8.
- Campisi J. Aging, cellular senescence, and cancer. Annu Rev Physiol. 2013;75:685-705. doi: 10.1146/annurev-physiol-030212-183653. Epub 2012 Nov 8.
- Reza Fazlalizadeh added an answer:What anesthetic can I use to bleed rats/mice from the orbital plexus?I am interested in doing retro orbital bleeding of mice/rats for doing FACS analysis of the whole blood. I am looking at a rare population. Right now I am using chloroform to do the same but I am not sure whether chloroform affects the cell numbers. Please tell me if chloroform influences the cell counts or any other parameter in the rodents and also suggest alternate and more reliable anesthetics.
You can find your answer here: http://web.jhu.edu/animalcare/procedures/retro-orbital.htmlA.
Anesthesia is required for retro-orbital bleeding.The following anesthetic agents are recommended for this procedure:
Mice - Ketamine (90 mg/kg) and Xylazine (10 mg/kg), IP
Pentobarbital (50mg/kg), IP
Proparacaine (Ophthetic®) - 1 drop per eye
Isoflurane (Drop Method) - contact vet staff
Rats - Ketamine (75 mg/kg) and Xylazine 10 mg/kg), IP
Pentobarbital (50 mg/kg), IP
Topical anesthetic, alone, may only be used in mice. Rats, who require more restraint due to their size, must be bled under general anesthesia. Note that there is the potential for contamination of the blood sample if topical anesthetic is used.
Use of any other anesthetic agents must be identified in the IACUC application.Following
- Carlo Palesi added an answer:Does chlorhexadine affect the viability of thawed stem cells?
Exploring a new catheter which is coated with chlorhexidine for stem cells infusion.
- Michael ER Olausson added an answer:What is the difference between cell engraftment and repopulation?
What is the difference between cell engraftment and repopulation?
I do not see the difference. If you have a malfunctioning tissue it can either be caused by cells missing due to specific targeted destruction, or a by a general destruction. If you believe that remaining signaling proteins in the ECM could "home" the cells needed, there is really no difference. The "homing" signal comes from the matrix proteins, either called repopulation or engraftment. If we want to differentiate between these entities, I would define engraftment the process of producing more than the original blueprint.Following
- Julie Piccand added an answer:EndoC-βH1 human pancreatic beta cell line.I want to use this cell line as a T2DM model. Who are the suppliers of this cell line in India?Following
- Bhagat Patlolla added an answer:Is it more efficient to induce the normal cells directly to the desired cells or induce them first to pluripotent then to desired cells?
These days some normal cells like fibroblast can also be transformed to desired cells type like neuron or cardiac myocytes or blood cell progenitors or other cells instead of inducing pluripotent stem cells then inducing them to differentiate to these cells. The question arises, why do we need to use stem cells rather than use direct transformation techniques. The efficiency and other things can be improved later. Please add your comments! Thanks!
I agree with you all. Tho Direct Differentiation is an attractive method for generating the target cells of interest, currently this technique is limited only to a few cell types. The challenge tho is getting more number of target cells(efficiency) and avoiding Dedifferentiation and Transdifferentiation of the target cells. Also, most importantly, the method of differentiation (non-viral to be precise).Following
- Soeren Turan added an answer:Will human stem cells grow alone (single cell clones in 96well)?
What considerations should I take to ensure a colony will grow from a single plated cell. i.e. should I add supernatent from a confulent colony and do I need to co-culture with other cells? Is there a specific media reagent that will promote growth in an isolated environment? Thanks.
Hi Sergey, is there any easier way to create the dialyzed albumin/medium solution ?
Why is albumin so important? Which albumin should we order (cat no?)
when i plate a 96 well plate with matrigel and rock, how many single clones can i expect, how many can i expect with laminin521?
- Holger Fey added an answer:How can I separate mononuclear cells from cord blood apart from ficoll hypaque?
There are so many papers stating that ficoll hypaque gradient can separate mononuclear cells from cord blood but is there any method apart from this? Kindly share detailed protocol.
Hmm, why would you want to use something different than a Ficoll density gradient to enrich MNCs? The method has been around for ages and there must be tens of thousands of labs that use it routinely. I've used it and it works every time. Take your cord blood and dilute it with an equal volume of 1 x PBS. Then layer the blood on 12 ml of Ficoll in a 50 ml conical tube and spin w/o brake at 2200 to 2400 rpm (depending on the centrifuge and rotor dimensions) for 25 minutes. You could use Percoll instead, but it's more or less the same thing, just a differnt compound.Following
- Younes BOUALLEGUI added an answer:What drives to predict the signaling cascade in response of a particular stimuli?I just want to know how to predict the response of a cell that activated a signalling event. I wish to discover any fundamental or computational tool to predict the signalling events.
If any exist, by giving the search phrase to the web to predict the possible mechanisms at dependent experimental conditions?
Best regards to thomas, realy he didn't let anything to say, very good sayed, i absolutely agree with him.Following
- Mandana Daneshvar added an answer:Does anyone have experience with stem cell growing?
I got embryonic stem cells 1 month ago and cultured them according to the protocol from its company. After 3 X making them passage, they start growing very slowly and not confluent well as we always expect. Everything is optimized and it shouldn't be existed any problem but this time I face to the problem. Does anyone know how I can overcome to the problem or have any suggestion to improve cells growing. I should mention that I use cell medium from company for seeding and growing the cells as well which needs to keep pluripotency of cells . I would be appreciated if anyone give me a clue to overcome the problem.
Thank you for all useful tips that you gave me. Now, It's working out and the cells start growing well. It supposed that it was caused by supplement from company because we used first seeding cells on supplement from company and then changing in differentiated medium for transplantation. Supplemets from company doesn't include many feeders for keeping pluripotency that's why they were growing slowly and I was worried to decline cell number on plate and also growing time but solved it now.Following
- Venil N Sumantran added an answer:How do I reduce cell proliferation while performing wound healing assay?Note: I tried low serum, however, my cells clump under low serum condition. Is there any other way?
Jiang, I have not done this assay myself. So, its better if you get protocol by reading literature.Following
- Joe Graymer added an answer:Aneuploidy in cancer "stem cells"?I have a question concerning aneuploidy in cancer "stem cells".
In most tumors one could observe cells unevenly divide their genetic material and producing aneuploid cells but in most "normal" cells aneuploidy is lethal. I know that there are some mutations allowing cancer cells to survive mild changes in chromosome composition but I believe that aneuploidy that goes too far must be lethal to cancer cells as well. So there should be some kind of balance - mutations increasing fitness but not to much. (so I believe most cases of hugely uneven genetic material composition that we can observe in microscopic analysis of tumors is only a by-product and these cells die just after division).
But how it is in case of cancer "stem cells"? Are they similarly aneuploidic as other "somatic" cancer cells or keep their chromosomes more under control?
You may like this:
- Sabine Siegemund added an answer:Phosflow technique to study signaling in hematopoietic stem cells?I want to study intracellular signaling in mouse hematopoietic stem/progenitor cells. Due to the fact that stem/progenitor cells are a very small population, it would be cool to use phosflow (detection by FACS) - especially the combination with surface marker staining would be very interesting for me ! Does anybody have good protocols - especially for hematopoietic cells?Following
- David Ebertz added an answer:Can anyone help with mammosphere quantification?I'm having trouble quantifying the weekly results of my mammosphere protocol for measuring stemness in cancer cell lines. Using StemCell's protocol, on the day of counting I:
harvest the mammosphere + media
Resuspend in 500uL of mmaosphere media
Transfer 50uL to a 96 well and count the numbers in this dilution
The main problem is that when I get to the 96 well, I've been seeing quite a reduction in the size of my mammospheres. I'm worried that the slight manipulation of the spheres leading up to this are breaking them up. But its also impossible to count if I were to keep the spheres in their normal 6-wells.
Does anyone have any experience or know people who have somewhat mastered mammospheres and could help me figure out how to better quantify? I could really use some help.
So I tried coating my own plate with (mammosphere media + 1.5% agarose). I used 70uL per well of a 96 well plate. I let it dry over night for good measure before seeding. The next day, I seeded cells for mammospher formation with 100uL of media per well. However, the very next day, all the cells were in the center of the dish. It was as they all moved towards the center even though I had laid down the agar mixture.
Has any of you had this problem before? Do I need to use more than 70uL to coat? Is it the wrong % of agar?
- Agnes Forsthuber added an answer:What are the chances that CMV promoter-lead genes in a lentivirus vector will undergo silencing after infecting stem cells?There are couple papers showing CMV is not an optimal promoter for creating stable cell lines from stem cells since this promoter and associated genes have high rate of silencing after transfection/transduction. My goal is to create a lentiviral clone with a non-CMV promoter to transfect stem cells. I have my gene in the gateway entry vector with CMV promoter and Life technologies do not have a lentiviral destination vector with EF1alpha or PGK promoter.
Can I easily excise the gene from the gateway entry vector and reclone it in other lentiviral vectors (non-gateway) with any promoter of my choice?
besides expression in stem cells: I am facing a similar problem in murin melanoma cells.
I successfully transfected murine melanoma cells (B16) with a lentiviral plasmid 5´LTR-CMV-GOI-SV40-ReportergeneRFP/Selectionmarker-3´LTR.
GOI - coding region of my GOI has 350bp and an n-terminal signal sequence and should be secreted.
I find reporter gene expression but no expression of GOI!
Could you maybe share a link of those ublications where the problems with CMV promotor are discussed?
I case of murine melanoma cells-could you suggest any other strong promotor that is consitituively acitve?Following
About Stem Cell Biology
All about stem cell biology! Stem Cell Protocols and Methods.