Stem Cell Method Discussion http://www.protocol-online.org/forums Thu, 09 Feb 2012 04:47:44 +0000 360 petri dishes vs tissue culture plates to grow BMDM http://www.protocol-online.org/forums/topic/24349-petri-dishes-vs-tissue-culture-plates-to-grow-bmdm/
I am trying to harvest the bone marrow and induce the growth of BMDM. However, I have came across many protocols that keep emphasizing the use of non-tissue culture plates (bacterial petri dishes) instead of tissue culture plates to grow them, while other seem to be ok using tissue culture plates.

What difference does it make if I use tissue culture plates instead of the non-tissue culture plate? Is this to eliminate the adherence of fibroblasts or DC ? isnt the addition of m-CSF is good enough to ensure only macrophages will grow by the end of the 6th day? any suggestions on what to use??]]> Thu, 09 Feb 2012 04:47:44 +0000 http://www.protocol-online.org/forums/topic/24349-petri-dishes-vs-tissue-culture-plates-to-grow-bmdm/ Spheres adhere in non-adherent conditions http://www.protocol-online.org/forums/topic/24119-spheres-adhere-in-non-adherent-conditions/
I am facing a problem which a cannot explain and I would very much appreciate your help.

I am culturing putative stem cells in non-adherent condiiton to grow as spheres.
I successfully obtained spheres which I was expanding. However, all of the sudden I started to have these circular strucutures at the bottom of the low attachemnt culture flask.
It resembles an adheren culture.

What I believe happen is that a shpere started to adhere and formed a circular monolayer attached culture.

Has anyone came across this problem and knows how to explain the phenomenon?

Thank you for your help.

Cheers.]]> Mon, 23 Jan 2012 11:25:49 +0000 http://www.protocol-online.org/forums/topic/24119-spheres-adhere-in-non-adherent-conditions/ sphere formation assay - pancreas adult stem cells http://www.protocol-online.org/forums/topic/23691-sphere-formation-assay-pancreas-adult-stem-cells/
I am trying to culture stem like cells from adult pancreas but i think something is missing in my media.
I am using the well described method for growing neural stem cells. hyclone dmem without serum, fgf, egf, heparin, n2, b27, neuro basal media and in low attachemnt conditions. does anyone know another strategy to culture adult pancreatic stem cells?

Cheers]]> Wed, 07 Dec 2011 15:22:51 +0000 http://www.protocol-online.org/forums/topic/23691-sphere-formation-assay-pancreas-adult-stem-cells/ how to form a pellet in pellet culture of MSC http://www.protocol-online.org/forums/topic/23424-how-to-form-a-pellet-in-pellet-culture-of-msc/

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]]> Tue, 15 Nov 2011 08:03:55 +0000 http://www.protocol-online.org/forums/topic/23424-how-to-form-a-pellet-in-pellet-culture-of-msc/ problem with dental pulp cell http://www.protocol-online.org/forums/topic/23284-problem-with-dental-pulp-cell/
I working with dental pulp cell for the isolation of stem cell, but in primary culture for one day to another cells die, in the culture no appearance of fungal. bacterial contamination. Also in other culture use trypsin to remove dead cell resulting in some tissue cells outgrowth (i do not use enzyme in the primary culture)

I need help]]> Tue, 01 Nov 2011 03:20:13 +0000 http://www.protocol-online.org/forums/topic/23284-problem-with-dental-pulp-cell/ Help needed- EBs were too small for differentiation http://www.protocol-online.org/forums/topic/23073-help-needed-ebs-were-too-small-for-differentiation/
My task was to form EB from mouse ipsc (2A-EGFPTg-3F) and from there, someone will use the EBs to differentiate to hematopoeitic cells. But, when we checked it, EBs were formed but they were too small, too small that they couldnt be used for differentiation.

What could have been the cause for this? and how can I improve it?

Thanks a lot!]]> Wed, 12 Oct 2011 13:09:16 +0000 http://www.protocol-online.org/forums/topic/23073-help-needed-ebs-were-too-small-for-differentiation/ NIH 3T3 Stable Transfection of Cell Line http://www.protocol-online.org/forums/topic/22936-nih-3t3-stable-transfection-of-cell-line/
This is my first post, so I'll give some background.

I have been trying to establish a stable transfected cell line of NIH 3T3. At first it appearred successful, I used G418S antibiotic for cell selection. A week post transfection and selection and a very small black rod shaped substance began attaching to the otherwise healthy looking cells. The rods appeared like a beard around the cells. At first I believed it to be cell waste and the normal cell death that occurs with selection however after an additional week the black rods are multiplying and sufocating the remaining healthy cells. The cells no longer reproduce and do not adhere as they should. What ever this is, it is not killed by pen/ strep or the G418S. Also, this substance does not change the media or completely kill the cells. I am now at week 4 and don't want to give up on these cells. Does any one have any ideas ?]]> Thu, 29 Sep 2011 12:36:22 +0000 http://www.protocol-online.org/forums/topic/22936-nih-3t3-stable-transfection-of-cell-line/ Help with forcing overexpression in hES cells http://www.protocol-online.org/forums/topic/22689-help-with-forcing-overexpression-in-hes-cells/
I'm a final year PhD student working on overexpressing a few GOIs in human ES cells. Up to now I have good data however I am missing one key thing - overexpression of a gene that I've been trying to do for a year now.

I'm overexpressing with the pCAG constitutive expression vector. I've managed to express GFP using this vector as a control, and one GOI that worked well. Sadly, another GOI is refusing to overexpress and I have no idea why.

I've sequenced the full vector with genscript and the vector sequence is 100% correct. So, it's not the DNA sequence that's the problem.

My cells are surviving in selection and so definitely have the vector in. When I come to do RT-qPCR for my GOI I get absolutely no overexpression over basal levels. I don't think my PCR is a problem. I've tried various Applied Biosystems Assay on Demands, and also designed my own primer probe mixes. Nothing.

Are there any companies out there that help with overexpressing genes for a cost? Or does anybody else have any idea what might be going on here? The GOI I previously managed to overexpress well is overexpressing 1800x over basal levels. That's huge. As such, I have no idea why my other gene isn't working.

Thanks for any ideas you may have. I could do with getting this working as it was supposed to be a whole chapter of my thesis based on the overexpression of this gene :s !!

Cheers!]]> Fri, 02 Sep 2011 23:39:18 +0000 http://www.protocol-online.org/forums/topic/22689-help-with-forcing-overexpression-in-hes-cells/ mouse bone marrow MSC culture http://www.protocol-online.org/forums/topic/22276-mouse-bone-marrow-msc-culture/ i am culturing the bone marrow cells of balb/c mice of 6-8 weeks. after 3 days of plating mesenchymal cells start appear(fibroblast like). But the MSCs do not multiply and after 14-15 days of culture, those cells die. I use DMEM+L-glutamine media. Please suggest me about how to get a confluent culture. Tue, 26 Jul 2011 13:08:15 +0000 http://www.protocol-online.org/forums/topic/22276-mouse-bone-marrow-msc-culture/ adult stem cell reporter cell line? http://www.protocol-online.org/forums/topic/22235-adult-stem-cell-reporter-cell-line/ I want to make cell lines which can mark adult stem cells, do you guys think it is worth to do it in mouse embryonic stem cells? is cell line like sox9, lgr5, and foxl1 reporter is going to be useful to track the progenitors? what about human embryonic stem cells can I use it for directed differentiation? anyone working on liver stem cells? will it be very difficult? any known culture condition? a lot questions, thank you guys Sat, 23 Jul 2011 04:21:33 +0000 http://www.protocol-online.org/forums/topic/22235-adult-stem-cell-reporter-cell-line/ How to prevent EB becoming too large http://www.protocol-online.org/forums/topic/21713-how-to-prevent-eb-becoming-too-large/
I am using mouse ES cells to form floating EB for 8 days to promote neuronal differentiation (Following 2004 Nature Neuroscience Paper, Volume 7 Number 9 Page 1003). My problem is that the floating EB, after 6-8 days in 10 cm petri dish in floating formation, become large and very dense in the center, presumably starting to die.
I do not have shaker in the incubator...Is there a good way to prevent EB becoming too big? Low density? Or shall I split the cells into multiple dishes?]]> Thu, 16 Jun 2011 18:07:02 +0000 http://www.protocol-online.org/forums/topic/21713-how-to-prevent-eb-becoming-too-large/ <![CDATA[CFSE doesn't work with HSC in 7 Day culture]]> http://www.protocol-online.org/forums/topic/21699-cfse-doesnt-work-with-hsc-in-7-day-culture/
I am using CFSE staining for freshly isolated hematopoietic stem cells (HSC) to track cell divisions/proliferation. The problem is, after 7 days of culture I cannot see distinct peaks for the genereations.
You can compare the result here:

http://dl.dropbox.com/u/14232908/CFSE%20Day%200%20and%20Day%207%20HSC.pptx

So there are several possible reasons for this result. Obviously, the staining itself worked, because the peak is going to the left. But the cell divisions are interfering with each other. So I might have seeded the cells in a too high density? There is no description that CFSE should not work for a high cell number.

Has anybody experience with CFSE?

Thanks
Cerise]]> Thu, 16 Jun 2011 08:36:04 +0000 http://www.protocol-online.org/forums/topic/21699-cfse-doesnt-work-with-hsc-in-7-day-culture/ EB too confluent? http://www.protocol-online.org/forums/topic/21621-eb-too-confluent/
I am following 2004 Nature Neuroscience paper to make neurons ( Volume 7 Number 9 Page 1003 "Differentiation of mouse embryonic stem cells into a defined neuronal lineage").

The attached picture is EB after 7 days, 4 days in ES medium and 3 days in ES + 5 uM RA. Does it look too confluent? They look very dark under scope and aggregate into big clumps.

Thanks

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]]> Mon, 13 Jun 2011 19:03:53 +0000 http://www.protocol-online.org/forums/topic/21621-eb-too-confluent/ Osteogenic Induction of MSCs, keep peeling off http://www.protocol-online.org/forums/topic/21573-osteogenic-induction-of-mscs-keep-peeling-off/
I seed my MSCs at 1100 cells/cm2 in 24 well plate formats and induce osteogenesis (medium formulation: 0.1nM Dexamethasone, 50nM Ascorbic Acid, 10mM Beta Glycerophosphate, high glucose DMEM, 10%FBS, 1% PenStrep) and change medium twice a week, for 3 and 4 weeks, but find that most times I try this, the cells begin to peel off 2 1/2 weeks into induction. By the time the 4week time point comes along, the cells are 90% gone, and I can't do nice alizarin red stainings at all. Anybody have any idea why this is happening to my cells?

Thanks!]]> Fri, 10 Jun 2011 05:00:54 +0000 http://www.protocol-online.org/forums/topic/21573-osteogenic-induction-of-mscs-keep-peeling-off/ MSC in culture - stop growing? http://www.protocol-online.org/forums/topic/21524-msc-in-culture-stop-growing/ This has happened at various passage number, it has happened at pass 5, 10, and recently transduced cells (GFP+) at pass 23 stopped expanding.
Does anyone have experience with this? What could be causing it, and can I kick start them again?
Thanks!]]> Tue, 07 Jun 2011 16:14:33 +0000 http://www.protocol-online.org/forums/topic/21524-msc-in-culture-stop-growing/ Stem Cells Sphere http://www.protocol-online.org/forums/topic/21391-stem-cells-sphere/
i would highly appreciate if somebody drops his reply here.

Thanks.]]> Tue, 31 May 2011 06:32:41 +0000 http://www.protocol-online.org/forums/topic/21391-stem-cells-sphere/ Inverse correlation in Cell Culture Treatment? http://www.protocol-online.org/forums/topic/21362-inverse-correlation-in-cell-culture-treatment/
Hi, I would like to ask regarding inverse correlation observed in cell culture treatment. One of the factor I use was EGF for differentiation purpose, the more concentrated I put in the media the lesser the number of cells. I wonder whether it's because of toxicity or it induce the cells to differentiate toward terminal and die off. I do believe EGF is a friendly growth factor, so I doubt the cytotoxicity and the fact that I only dilute using PBS plus BSA. Anyone experience using EGF in cell culture that could share opinion? :)

Thank you so much and I appreciate all the attention. :)

Best Regards,

Nani]]> Mon, 30 May 2011 01:01:34 +0000 http://www.protocol-online.org/forums/topic/21362-inverse-correlation-in-cell-culture-treatment/ iPSC http://www.protocol-online.org/forums/topic/21100-ipsc/
I'm trying to make iPSCs using the lentiviral method, but after 10 times of trying, I cant get anything.. not a single colony appears. anyone with experience on what to look out for?


Thanks a lot!!!]]> Mon, 16 May 2011 09:16:36 +0000 http://www.protocol-online.org/forums/topic/21100-ipsc/ Inducing apoptosis in ESCs by UV http://www.protocol-online.org/forums/topic/20968-inducing-apoptosis-in-escs-by-uv/
Anyone have any experience inducing apoptosis in ES cells, in terms of how much UV exposure to give and how long to wait afterwards? I was thinking ballpark 50J/m2, followed by overnight 12-16 hour incubation..]]> Mon, 09 May 2011 17:03:11 +0000 http://www.protocol-online.org/forums/topic/20968-inducing-apoptosis-in-escs-by-uv/ MEF not attached! http://www.protocol-online.org/forums/topic/20859-mef-not-attached/
My mef didnt attached to the well after irradiation. They were seeded onto a 4-well plate(1.9cm2/well). The wells were coated with gelatin before. Viability of 95% was tested with Trypan blue. Any of you can give me suggestion.]]> Tue, 03 May 2011 01:28:00 +0000 http://www.protocol-online.org/forums/topic/20859-mef-not-attached/