All about stem cell biology! Stem Cell Protocols and Methods.

• Beiyan Zhou

  Recently, more and more people are using ALDH staining method to establish "stemness" of subpopulations in either cell lines or primary cells. The major question for me is how to interoperate theRecently, more and more people are using ALDH staining method to establish "stemness" of subpopulations in either cell lines or primary cells. The major question for me is how to interoperate the ALDH results. In general, I think this is a method with arbitrary setting based on the higher or even higher enzymatic activities, more or even more abundance of an enzyme, that believe should be linked with “stemness”. The gating is always very subjective: no distinct population (which can be true for heterogeneity of cells with various activities or abundance aldehyde dehydrogenase). From ours and others results, it can be used as a way to demonstrate higher detoxification related enzymatic ability-->”assumption”: usually higher in stem/progenitor cells--->thus, a guide to enrich stem/progenitor cell subpopulations. More or less, it resembles the method of "side population" which is a alternative method to establish "stemness" with "detoxification ability". However, in the cancer or cancer stem cell (especially using cell lines), the two aspects may still linked but with much less power to enrich for “stemness” for that one of the major property of cancer is enhance survival against drug, immune cell attack, environmental harm, etc. I think about how to interpret the population with relatively high ALDH population, especially how much it Truly represents pertaining to cell properties and "stemness" related cell identities, we should take extra precaution. Thank you in advance to sharing your thoughts with me!

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    Beiyan Zhou replied

    Dear Alasdair, Thank you very much for sharing your thoughts with me! I am looking forward to see your new report on this issue!

    10 days ago
• Sumia Mohamed Essid

  Amino acid transport

  Does any one have a protocol to measure the amino acid transport in muscle cell line
• Signe Diness Vindeløv

  How do you analyse FACS data obtained by the ALDEFLUOR kit?

  How do you analyse the FACS data obtained by the ALDEFLUOR kit? Where do you set the gate on the inhibited sample (DEAB)? What should be the cut-off on the inhibited sample? If you set the ALDHhiHow do you analyse the FACS data obtained by the ALDEFLUOR kit? Where do you set the gate on the inhibited sample (DEAB)? What should be the cut-off on the inhibited sample? If you set the ALDHhi gate really close to the cell popluation in the DEAB sample then you have a rather high intra-experimental variation. Look forward to hear from anybody who worked with this kit.

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    Hazem Ghebeh replied

    For me your intra-experimental variations are most likely to due to preparation of cells and ALDH staining and not due to gating.. Standardizing the time, confluence level and state of cells willFor me your intra-experimental variations are most likely to due to preparation of cells and ALDH staining and not due to gating.. Standardizing the time, confluence level and state of cells will affect a lot the ALDH staining..etc. Important to use FSC-A versus FSC-W to remove douplets Thanks,

    26 days ago
• Zhengqiang Yuan

  MSC culture, 10% or 20% FCS?

  Hello, guys, Is 10% or 20% FCS better for the culture of mesenchymal stem cells, please? I need to expand a lot of MSC for in vivo experiments. Am struggling for this. Many thanks, Qiang

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    Wayne Thomas replied

    The optimal media we found after CFU-F comparison testing is alphMEM with low glucose and 16% FBS (Gibco -MSC qualified). To start the primary culture off you may add human serum. The quantity ofThe optimal media we found after CFU-F comparison testing is alphMEM with low glucose and 16% FBS (Gibco -MSC qualified). To start the primary culture off you may add human serum. The quantity of FBS 10-20% will just slow or speed up the growth rates.

    29 days ago
• Chiranjeevi Sandi

  Does anyone have a good protocol to differentiate the Neural Stem Cells (NSCs) into neurons?

  I used RA based method on adult NSCs and obtained less percentage of neurons. However, when I use the foetal NSCs I got slightly improved number of neurons but not a significant increase. For NSCI used RA based method on adult NSCs and obtained less percentage of neurons. However, when I use the foetal NSCs I got slightly improved number of neurons but not a significant increase. For NSC differentiation, I am using NSC basal medium + B27 supplements + RA (10μM) for first five days followed by changing the medium to NSC basal medium + B27 supplements + BDNF for next 5-10days (maturation of neurons). Any suggestions of using different medium or reagents to increase the neuron population are greatly appreciated. Thanks, Chiru

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    Johnathan Nuttall replied

    If you inhibit mitosis only neurons will survive. For example 1 uM cytosine-h-d- arabinofuranoside should work.

    Apr 8, 2012
• Mahmood Talkhabi

  Can anyone tell me how to determine and isolate the cardiac progenitors during differentiation of mouse embryonic stem cell into cardiomyocyte?

  I want to determine and then isolate different types of cardiac progenitor during differentiation of mESCs and need a simple method to do that.

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    Fatemeh Babaei replied

    Did you find your answer,this is my question too!!

    Apr 7, 2012
• Benny Burn

  Neural Stem Cells (NSCs) and Embryonic Stem cells (ESCs)

  Do NSCs form colonies like ESCs do in vitro? I seldom hear people mentioning NSC colony. Are NSCs colony as tightly packed as cells in ESCs colony? So when passaging NSCs colony, should they beDo NSCs form colonies like ESCs do in vitro? I seldom hear people mentioning NSC colony. Are NSCs colony as tightly packed as cells in ESCs colony? So when passaging NSCs colony, should they be kept as small clusters so as to prevent NScs from differentiating? I've grown RenVM (ReNeuron VM) cells before and they do not form colonies in vitro.

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    Sharona Even-Ram replied

    The term NSC can refer to different things: cells that are derived from embryonic , newborn or adult brains at different ages or cells derived from ESC that are directed to differentiate into neuralThe term NSC can refer to different things: cells that are derived from embryonic , newborn or adult brains at different ages or cells derived from ESC that are directed to differentiate into neural lineages. Which one is yours? NSCs (both types) definitely form colonies and it is a well known phenomenon. Quite often they form a "flagship" morphology, where neurites project from the spheric colony and connect to other colonies. Cells in the NSC colonies are usually much smaller then ESC cells. It is hard to keep the cells from differentiating, especially in spherical colonies where cells change according to their topology. There are many ways to passage NSCs and they vary from mechanical dissociation to enzymatic or chemical methods. You really need to define better the purpose if you want a more accurate answer.

    Apr 5, 2012
• Benny Burn

  Reasons for centre of ESC colony dying?

  Why, when ESCs colony grows too big, would the cells in the centre start dying?

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    Sharona Even-Ram replied

    As some suggested here, it might be due to insufficient oxygen and nutrient supply, however, centers of ESC colonies normally do not reach the limiting size that occurs in spheroids. In my hands,As some suggested here, it might be due to insufficient oxygen and nutrient supply, however, centers of ESC colonies normally do not reach the limiting size that occurs in spheroids. In my hands, even if the center is still relatively 'flat' cells would start dying. A possible explanation is that cells at the center that keep proliferating have no space to attach to (as opposed to cells in the margins). Except for cells at the basal layer, the other cells have 2 options: to develop cell-cell contacts instead of cell-matrix ones, or to undergo apoptosis. In your case the second option seems to prevail.

    Apr 5, 2012
• David Rhoads

  Is there a consistent moderate-throughput transient transfection technique for promoter analyses in mouse ES cells?

  Currently, the ES cells are maintained on a MEF feeder layer but seeded onto gelatin alone in a 48-well cluster for transfection with Lipofectamine 2000. Electroporation or nucleofection would be tooCurrently, the ES cells are maintained on a MEF feeder layer but seeded onto gelatin alone in a 48-well cluster for transfection with Lipofectamine 2000. Electroporation or nucleofection would be too cumbersome. I haven't tested other transfection reagents yet or whether including MEFs for seeding would produce an unacceptable background.

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    Meera Nair replied

    There are many strategies used , there is a paper by Ko et al., 2008 which uses liposome-based transfection methods by introduction of a plasmid encoding enhanced green fluorescent protein (EGFP)There are many strategies used , there is a paper by Ko et al., 2008 which uses liposome-based transfection methods by introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into mouse ES (mES) cells. The weblink is http://www.nature.com/gt/journal/v16/n1/full/gt2008125a.html Please do have a look

    Mar 30, 2012
• Ariadni Papaja

  Can anyone recommend the antibody for the surface marker of mouse cardiomyocytes?

  I would like to do staining/flow cytometry of the mouse cardiomyocyte (adults and neonatal) without fixing the cells. Does anyone know about the surface marker for mouse cardiomyocytes? can youI would like to do staining/flow cytometry of the mouse cardiomyocyte (adults and neonatal) without fixing the cells. Does anyone know about the surface marker for mouse cardiomyocytes? can you recommend a good antibody for it? alternatively, is there any good protocol for staining cardiomyocytes (fixed) without disrupting/disturbing the membrane protein? thank you very much!
• Haoyang Jiang

  How to use Fluo-3 to evaluate the concentration of Calcium?

  Is there anyone who can recommend a protocol to evaluate the concentration of Calcium with Fluo-3? What should I be careful with?

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    Roberta Ribeiro Costa replied

    In order to evaluate the calcium concentration a ratiometric Ca indicator is more adequated.

    Mar 29, 2012
• Fatma Eyuboglu

  What do you prefer to use for understanding the migration of cells in vivo after transplantation?

  Other than GFP, are DAPI or Hoecst suitable for this? My aim is labeling the mesenchymal stem cells and, after 60 days, observing them from the tissue of recepient rat.

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    Katerina Anesti replied

    Thank you!

    Mar 22, 2012
• Ashaq husssain Najar

  Should/does B27 supplement used in the protocols for deriving neural precursors cells from mouse ES cells contain Vitamin A (retinoic acid) or not?

  There are many protocols out there which suggest the use of B27 supplement along with N2 supplement for efficient derivation of neural stem/precursor cells from mouse ES cells (based on AustinThere are many protocols out there which suggest the use of B27 supplement along with N2 supplement for efficient derivation of neural stem/precursor cells from mouse ES cells (based on Austin Smith's method). I am trying the same, but with no success. I m using B27 without vitamin A. To what an extent could the presence or absence of Vitamin A in the media make a difference in my case. Also could anybody suggest a good protocol for the conversion of mouse ES cells into neural precursors?

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    Ashaq husssain Najar replied

    yes Alexandra, i dont need any lineage-specific neural cells. using RA, what I fear is whether i wud b able to maintain the cells in precursor stage or not ...i also agree with Siddhartha in sayingyes Alexandra, i dont need any lineage-specific neural cells. using RA, what I fear is whether i wud b able to maintain the cells in precursor stage or not ...i also agree with Siddhartha in saying that something might be wrong with the cell line itself..anywayz thanks a lot to both of u for ur valualble inputs...i wud need ur suggestions in future as well....take care!

    Mar 21, 2012
• Margaret Clotworthy

  "EU bans embryonic stem cell patents but decision may have limited implications" http://www.nature.com/nrd/journal/vaop/ncurrent/full/nrd3612.html What do you think about this development? Best"EU bans embryonic stem cell patents but decision may have limited implications" http://www.nature.com/nrd/journal/vaop/ncurrent/full/nrd3612.html What do you think about this development? Best regards, Margaret, Dr Margaret Clotworthy, Director, Human Focused Testing www.humanfocusedtesting.com - for all your research needs

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    Beatriz Lacerda Sousa replied

    Besides the enormous barriers already in place in the european scientific community due to funding this will be barely noticed. The level of purely industrial and biotech research in Europe is stillBesides the enormous barriers already in place in the european scientific community due to funding this will be barely noticed. The level of purely industrial and biotech research in Europe is still marginal. Given the amount of money that is required to do stem cell research and the number of stem cell labs spread out through Europe the ban won't be noticed.

    Mar 12, 2012
• Pooja Vijaygopal

  Help with housekeeping gene

  Is there anyone who is studying mouse embryonic gonad development and knows a good house keeping gene?

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    Beatriz Lacerda Sousa replied

    we use the double system (so we normalize gene expression to 2 housekeeping genes: L7 and UBB)

    Mar 12, 2012